Tiglien-3-one derivatives

ABSTRACT

The present invention relates to tiglien-3-one compounds and their use in methods of treating or preventing protozoal infections, bacterial infections, parasitic infections and cell proliferative disorders. The tiglien-3-one compounds are also used in methods of controlling pests in humans, animals, plants and the environment

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/149,977 filed Oct. 2, 2018, which is a continuation of U.S.application Ser. No. 15/426,744 filed Feb. 7, 2017, which is acontinuation of U.S. application Ser. No. 15/041,960 filed Feb. 11,2016, now U.S. Pat. No. 9,770,431 issued Sep. 26, 2017, which is adivisional of U.S. application Ser. No. 14/084,949 filed Nov. 20, 2013,now U.S. Pat. No. 9,289,410 issued Mar. 22, 2016, which is a divisionalof U.S. application Ser. No. 12/158,461 filed Nov. 17, 2008, now U.S.Pat. No. 8,598,229 issued Dec. 3, 2013, a U.S. National Stage entry ofPCT/AU2006/002001 filed Dec. 22, 2006, which claims the benefit ofAustralian Patent Application 2005907278 filed Dec. 23, 2005, all ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to bioactive molecules. More particularly, thisinvention relates to tiglien-3-one derivatives of potential therapeuticbenefit and/or of use as a pharmaceutical and as an agrochemical.

BACKGROUND OF THE INVENTION

Bio-discovery is a growing field, which investigates and screens forbioactive natural products from natural environments, including plants,microorganisms, coral and other marine life. In the search for bioactivenatural products, biological material is screened for molecules havingproperties that may be of therapeutic benefit for potential use in arange of treatments, for example treatments for cancer, antiprotozoaltreatments, antiparasitic treatments, antibiotic treatments andanti-inflammatory treatments, or for pesticidal activity.

SUMMARY OF THE INVENTION

The present invention arises from the discovery of new tiglien-3-onederivatives which have potentially new therapeutic uses as cytotoxicagents, antiprotozoal agents, antiparasitic agents and antibiotic agentsor potential as pesticidal agents for agricultural use.

One aspect of the invention provides compounds of the formula I

wherein:

X is selected from —S—, —O—, —NH— or —N(C₁₋₆ alkyl)-;

each n is independently selected from 1 to 10;

R¹ to R²⁴ are each independently selected from hydrogen, —C₁-C₂₀ alkyl,—C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, —C₃-C₈ cycloalkyl, —C₆-C₁₄ aryl,—C₅-C₁₄ heteroaryl, —C₃-C₁₄ heterocyclyl, arylalkyl, heteroarylalkyl,heterocyclylalkyl, halo, —CN, —NO₂, —C₁-C₁₀ haloalkyl, —C₁-C₁₀dihaloalkyl, —C₁-C₁₀ trihaloalkyl, —COR²⁸, —CO₂R²⁸, —OR²⁸, —SR²⁸,—N(R²⁸)₂, —NR²⁸OR²⁸, —ON(R²⁸)₂, —SOR²⁸, —SO₂R²⁸, —SO₃R²⁸, —SON(R²⁸)₂,—SO₂N(R²⁸)₂, —SO₃N(R²⁸)₂, —P(R²⁸)₃, —P(O)(R²⁸)₃, —OSi(R²⁸)₃, —OB(R²⁸)₂,—C(Z)R²⁸ and —ZC(Z)R²⁸;

R²⁸ is selected from hydrogen, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀alkynyl, —C₃-C₈ cycloalkyl, —C₆-C₁₄ aryl, —C₅-C₁₄ heteroaryl, —C₃-C₁₄heterocyclyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, —C₁-C₁₀haloalkyl, —C₁-C₁₀ dihaloalkyl and —C₁-C₁₀ trihaloalkyl; or

one or more of R¹ (or R²) is connected to R³ (or R⁴), and/or R³ (or R⁴)is connected to R⁵ (or R⁶), and/or R⁵ (or R⁶) is connected to R⁷, and/orR⁷ is connected to R⁸ (or R⁹), and/or R⁸ (or R⁹) is connected to R¹⁰ (orR¹¹), and/or R¹⁰ (or R¹¹) is connected to R¹², and/or R¹² is connectedto R¹³ (or R¹⁴), and/or R¹³ is connected to R¹⁴, and/or R¹³ (or R¹⁴) isconnected to R¹⁵, and/or R¹⁵ is connected to R¹⁷, and/or R¹⁸ (or R¹⁹ orR²⁰) is connected to R²² (or R²¹), and/or R²² (or R²¹) is connected toR²³, and/or R²³ is connected to R¹ (or R²), and/or R²⁴ is connected toR⁷ (or R¹⁶) or R⁸ (or R⁹) to form a —C₁-C₈ disubstituted (fused)saturated or unsaturated carbo- and heterocyclic rings furthersubstituted by R²⁸, —(C═Z)R²⁸ and —Z(C═Z)R²⁸; or

one or more of R¹ (or R²) is connected to R³ (or R⁴), R³ (or R⁴) isconnected to R⁵ (or R⁶), R⁵ (or R⁶) is connected to R⁷, R⁷ is connectedto R²⁴, R⁸ (or R⁹) is connected to R¹⁰ (or R¹¹), R¹⁶ is connected toR²⁴, R²² (or R²¹) is connected to R²³, R²³ is connected to R¹ (or R²),R²⁴ is connected to R⁷ (or R¹⁶) or R⁸ (or R⁹) to form a double bond, anepoxide or a thioepoxide; or

one or more of R¹ and R², R³ and R⁴, R⁵ and R⁶, R⁸ and R⁹, R¹⁰ and R¹¹,R¹⁸ and R¹⁹ (or R¹⁸ and R²⁰), R²¹ and R²² form a double bond to Z, and

Z is selected from sulfur, oxygen and —NH— or —N(C₁-C₆ alkyl)-;

or a pharmaceutically, agriculturally or pesticidally acceptable saltthereof.

In some embodiments, when any one or more of R¹ to R²⁴ is —C₂-C₂₀alkenyl, the alkenyl units may be singular, multiple, allenyl and/orconjugated or skipped.

In other embodiments, where any one or more of R¹ to R¹⁰ is —C₂-C₂₀alkynyl, one or more of R¹ to R¹⁰ may further comprise an aryl orheteroaryl group.

In still other embodiments when any one or more of R¹ to R²⁴ is —C₂-C₂₀alkynyl, the alkynyl units may be singular or multiple alkynyl units.

In another aspect of the invention, there is provided a compound offormula II

wherein:

X is selected from —O—, —S— and —NR²⁵—;

R¹ and R² are independently selected from hydrogen, —OH, —OC₁-C₁₀ alkyl,—OC₂-C₁₀ alkenyl, —OC₂-C₁₀ alkynyl, —Ocycloalkyl, —OC(O)C₁-C₁₀ alkyl,—OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀ alkynyl, —OC(O)cycloalkyl,—OC(O)heterocyclyl, —OC(O)heteroaryl, —OC(O)NHC₁-C₁₀ alkyl,—OC(O)NHC₂-C₁₀ alkenyl, —OC(O)NHC₂-C₁₀ alkynyl, —OC(O)NHcycloalkyl,—OC(O)NHaryl, —OC(O)NHheterocyclyl, —OC(O)NHheteroaryl, —OC(S)NHC₁-C₁₀alkyl, —OC(S)NHC₂-C₁₀ alkenyl, —OC(S)NHC₂-C₁₀ alkynyl,—OC(S)NHcycloalkyl, —OC(S)NHaryl, —OC(S)NHheterocyclyl,—OC(S)NHheteroaryl or R¹ and R² taken together are ═O, ═S, ═NH or═N(C₁-C₆ alkyl);

R³ is hydrogen, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl or —C₂-C₁₀ alkynyl;

R⁴ and R⁵ are each hydrogen, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl, —C₂-C₁₀alkynyl, —OH, —OC₁-C₁₀ alkyl, —OC₂-C₁₀ alkenyl, —OC₂-C₁₀ alkynyl,—Ocycloalkyl, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀ alkenyl, —OC(O)C₂-C₁₀alkynyl, —OC(O)cycloalkyl, —OC(O)aryl, —OC(O)heterocyclyl,—OC(O)heteroaryl, —OC(O)NHC₁-C₁₀ alkyl, —OC(O)NHC₂-C₁₀ alkenyl,—OC(O)NHC₂-C₁₀ alkynyl, —OC(O)NHcycloalkyl, —OC(O)NHaryl,—OC(O)NHheterocyclyl, —OC (O)NHheteroaryl, —OC(S)NHC₁-C₁₀ alkyl,—OC(S)NHC₂-C₁₀ alkenyl, —OC(S)NHC₂-C₁₀ alkynyl, —OC(S)NHcycloalkyl,—OC(S)NHaryl, —OC(S)NHheterocyclyl, —OC(S)NHheteroaryl, F, Cl, Br, I,—CN, —NO₂ or N(R²⁵)₂, or R⁴ and R⁵ taken together form a double bond orare —O—, —S—, —NR²⁵— or —CR²⁶R²⁷—;

R⁶ is selected from hydrogen, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl and —C₂-C₁₀alkynyl;

R⁸ is selected from hydrogen, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl and —C₂-C₁₀alkynyl;

R¹⁰ is selected from hydrogen, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl and—C₂-C₁₀ alkynyl;

R¹¹ is selected from —OH, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl, —OC₂-C₂₀alkynyl, —Ocycloalkyl, —OC(O)C₁-C₂₀ alkyl, —OC(O)C₂-C₂₀ alkenyl,—OC(O)C₂-C₂₀ alkynyl, —OC(O)cycloalkyl, —OC(O)aryl, —OC(O)heterocyclyl,—OC(O)heteroaryl, —OC(O)NHC₁-C₂₀ alkyl, —OC(O)NHC₂-C₂₀ alkenyl,—OC(O)NHC₂-C₂₀ alkynyl, —OC(O)NHcycloalkyl, —OC(O)NHaryl,—OC(O)NHheterocyclyl, —OC(O)NHheteroaryl, —OC(S)NHC₁-C₂₀ alkyl,—OC(S)NHC₂-C₂₀ alkenyl, —OC(S)NHC₂-C₂₀ alkynyl, —OC(S)NHcycloalkyl,—OC(S)NHaryl, —OC(S)NHheterocyclyl, —OC(S)NHheteroaryl or R¹⁰ and R¹¹taken together form a carbonyl group (═O);

R¹² is selected from —OH, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl, —OC₂-C₂₀alkynyl, —Ocycloalkyl, —OC(O)C₁-C₂₀ alkyl, —OC(O)C₂-C₂₀ alkenyl,—OC(O)C₂-C₂₀ alkynyl, —OC(O)cycloalkyl, —OC(O)aryl, —OC(O)heterocyclyl,—OC(O)heteroaryl, —OC(O)NHC₁-C₂₀ alkyl, —OC(O)NHC₂-C₂₀ alkenyl,—OC(O)NHC₂-C₂₀ alkynyl, —OC(O)NHcycloalkyl, —OC(O)NHaryl,—OC(O)NHheterocyclyl, —OC(O)NHheteroaryl, —OC(S)NHC₁-C₂₀ alkyl,—OC(S)NHC₂-C₂₀ alkenyl, —OC(S)NHC₂-C₂₀ alkynyl, —OC(S)NHcycloalkyl,—OC(S)NHaryl, —OC(S)NHheterocyclyl, —OC(S)NHheteroaryl;

R¹³ and R¹⁴ are independently selected from hydrogen and —C₁-C₁₀ alkyl;

R¹⁸ is selected from C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl,—OH, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl, —OC₂-C₂₀ alkynyl, —Ocycloalkyl,—OC(O)C₁-C₂₀ alkyl, —OC(O)C₂-C₂₀ alkenyl, —OC(O)C₂-C₂₀ alkynyl,—OC(O)cycloalkyl, —OC(O)aryl, —OC(O)heterocyclyl, —OC(O)heteroaryl,—NHC₁-C₂₀ alkyl, —NHC₂-C₂₀ alkenyl, —NHC₂-C₂₀ alkynyl, —NHcycloalkyl,—NHaryl, —NHheterocyclyl, —NHheteroaryl, —OC(O)NHC₁-C₂₀ alkyl,—OC(O)NHC₂-C₂₀ alkenyl, —OC(O)NHC₂-C₂₀ alkynyl, —OC(O)NHcycloalkyl,—OC(O)NHaryl, —OC(O)NHheterocyclyl, —OC(O)NHheteroaryl, —OC (S)NHC₁-C₂₀alkyl, —OC(S)NHC₂-C₂₀ alkenyl, —OC(S)NHC₂-C₂₀ alkynyl,—OC(S)NHcycloalkyl, —OC(S)NHaryl, —OC(S)NHheterocyclyl,—OC(S)NHheteroaryl;

R¹⁹ and R²⁰ are independently selected from hydrogen, —C₁-C₆ alkyl,—C₂-C₆ alkenyl, —C₂-C₆ alkynyl, —OH, —OC₁-C₆ alkyl, —OC₂-C₆ alkenyl,—OC₂-C₆ alkynyl, —Ocycloalkyl, —OC(O)C₁-C₆ alkyl, —OC(O)C₂-C₆ alkenyl,—OC(O)C₂-C₆ alkynyl, —OC(O)cycloalkyl, —OC(O)aryl, —OC(O)heterocyclyl,—OC(O)heteroaryl or R¹⁹ and R²⁰ taken together form a carbonyl orthiocarbonyl group;

R²¹ is selected from hydrogen, —C₁-C₁₀ alkyl, —C₂-C₁₀ alkenyl and—C₂-C₁₀ alkynyl;

R²² is selected from hydrogen, —OH, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl,—OC₂-C₂₀ alkynyl, —Ocycloalkyl, —OC(O)C₁-C₂₀ alkyl, —OC(O)C₂-C₂₀alkenyl, —OC(O)C₂-C₂₀ alkynyl, —OC(O)cycloalkyl, —OC(O)aryl,—OC(O)heterocyclyl, —OC(O)heteroaryl, —OC(O)NHC₁-C₂₀ alkyl,—OC(O)NHC₂-C₂₀ alkenyl, —OC(O)NHC₂-C₂₀ alkynyl, —OC(O)NHcycloalkyl,—OC(O)NHaryl, —OC(O)NHheterocyclyl, —OC(O)NHheteroaryl, —OC(S)NHC₁-C₂₀alkyl, —OC(S)NHC₂-C₂₀ alkenyl, —OC(S)NHC₂-C₂₀ alkynyl,—OC(S)NHcycloalkyl, —OC(S)NHaryl, —OC(S)NHheterocyclyl,—OC(S)NHheteroaryl or R²¹ and R²² taken together form a carbonyl group;

R²³ is selected from hydrogen, —OH, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl,—OC₂-C₂₀ alkynyl, —Ocycloalkyl, —OC(O)C₁-C₂₀ alkyl, —OC(O)C₂-C₂₀alkenyl, —OC(O)C₂-C₂₀ alkynyl, —OC(O)cycloalkyl, —OC(O)aryl,—OC(O)heterocyclyl, —OC(O)heteroaryl, —OC(O)NHC₁-C₂₀ alkyl,—OC(O)NHC₂-C₂₀ alkenyl, —OC(O)NHC₂-C₂₀ alkynyl, —OC(O)NHcycloalkyl,—OC(O)NHaryl, —OC(O)NHheterocyclyl, —OC(O)NHheteroaryl, —OC(S)NHC₁-C₂₀alkyl, —OC(S)NHC₂-C₂₀ alkenyl, —OC(S)NHC₂-C₂₀ alkynyl,—OC(S)NHcycloalkyl, —OC(S)NHaryl, —OC(S)NHheterocyclyl,—OC(S)NHheteroaryl;

R²⁴ is selected from hydrogen, —OH, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl,—OC₂-C₂₀ alkynyl, —Ocycloalkyl, —OC(O)C₁-C₂₀ alkyl, —OC(O)C₂-C₂₀alkenyl, —OC(O)C₂-C₂₀ alkynyl, —OC(O)cycloalkyl, —OC(O)aryl,—OC(O)heterocyclyl, —OC(O)heteroaryl, —OC(O)NHC₁-C₂₀ alkyl,—OC(O)NHC₂-C₂₀ alkenyl, —OC(O)NHC₂-C₂₀ alkynyl, —OC(O)NHcycloalkyl,—OC(O)NHaryl, —OC(O)NHheterocyclyl, —OC(O)NHheteroaryl, —OC(S)NHC₁-C₂₀alkyl, —OC(S)NHC₂-C₂₀ alkenyl, —OC(S)NHC₂-C₂₀ alkynyl,—OC(S)NHcycloalkyl, —OC(S)NHaryl, —OC(S)NHheterocyclyl,—OC(S)NHheteroaryl;

R²⁵ is selected from hydrogen and —C₁-C₁₀ alkyl;

R²⁶ and R²⁷ are independently selected from hydrogen, —C₁-C₁₀ alkyl,—OH, —OC₁-C₁₀ alkyl;

wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl andheteroaryl is optionally substituted;

and geometric isomers or stereoisomers thereof and pharmaceutically,agriculturally or pesticidally acceptable salts thereof.

In one embodiment, the taglienone derivative of formula (II) is acompound of formula (III)

wherein:

R³ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl;

R⁴ and R⁵ are each independently hydrogen, —C₁-C₆ alkyl, —C₂-C₆ alkenyl,—OH, —OC₁-C₆ alkyl, —OC₂-C₆ alkenyl, —OC(O)C₁-C₆ alkyl, —OC(O)C₂-C₆alkenyl, —OC(O)aryl, —OC(O)heterocyclyl, —OC(O)heteroaryl, F, Cl, Br orI, or R⁴ and R⁵ taken together form a double bond or are —O—;

R⁶ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl;

R⁸ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl;

R¹³ and R¹⁴ are each independently hydrogen or —C₁-C₆ alkyl;

R^(a), R^(b), R^(c), R^(d), R^(e) and R^(f) are each independentlyselected from hydrogen, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl,—C(O)C₁-C₂₀ alkyl, —C(O)C₂-C₂₀ alkenyl, —C(O)C₂-C₂₀ alkynyl,—C(O)cycloalkyl, —C(O)aryl, —C(O)heterocyclyl, —C(O)heteroaryl,—C(O)NHC₁-C₂₀ alkyl, —C(O)NHC₂-C₂₀ alkenyl, —C(O)NHC₂-C₂₀ alkynyl,—C(O)NHcycloalkyl, —C(O)NHaryl, —C(O)NHheterocyclyl, —C(O)NHheteroaryl,—C(S)NHC₁-C₂₀ alkyl, —C(S)NHC₂-C₂₀ alkenyl, —C(S)NHC₂-C₂₀ alkynyl,—C(S)NHcycloalkyl, —C(S)NHaryl, —C(S)NHheterocyclyl and—C(S)NHheteroaryl;

wherein each alkyl, alkenyl, aryl, heterocyclyl and heteroaryl group isoptionally substituted;

or geometric isomers or stereoisomers and pharmaceutically,agriculturally or pesticidally acceptable salts thereof.

In a preferred embodiment, the compound of formula (II) is a compound offormula IV

wherein:

R³ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl;

R⁴ and R⁵ are each independently hydrogen, —C₁-C₆ alkyl, —C₂-C₆ alkenyl,—OH, —OC₁-C₆ alkyl, —OC₂-C₆ alkenyl, —OC(O)C₁-C₆ alkyl, —OC(O)C₂-C₆alkenyl, —OC(O)aryl, —OC(O)heterocyclyl, —OC(O)heteroaryl, F, Cl, Br orI, or R⁴ and R⁵ taken together form a double bond or are —O—;

R⁶ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl;

R⁸ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl;

R¹⁰ is hydrogen;

R¹¹ is hydroxy, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl, —OC(O)C₁-C₂₀ alkyl,—OC(O)C₂-C₂₀ alkenyl, —OC(O)aryl, —OC(O)heterocyclyl, —OC(O)heteroaryl;or R¹⁰ and R¹¹ taken together form a carbonyl group;

R¹²⁹ is hydrogen, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C(O)C₁-C₂₀ alkyl,—C(O)C₂-C₂₀ alkenyl, —C(O)aryl, —C(O)heterocyclyl or —C(O)heteroaryl;

R¹³ and R¹⁴are each independently hydrogen or —C₁-C₆ alkyl;

R¹⁸⁹ is hydrogen, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C(O)C₁-C₆ alkyl,—C(O)C₂-C₆ alkenyl, —C(O)aryl, —C(O)heterocyclyl or —C(O)heteroaryl;

R¹⁹ and R²⁰ ° are independently selected from hydrogen, —OH, —OC₁-C₆alkyl, —OC₂-C₆ alkenyl or R¹⁹ and R²⁰ taken together form a carbonylgroup;

R²¹ is hydrogen;

R²² is hydroxy, —OC₁-C₆ alkyl, —OC₂-C₆ alkenyl, —OC(O)C₁-C₆ alkyl,—OC(O)C₂-C₆ alkenyl, —OC(O)aryl, —OC(O)heterocyclyl, —OC(O)heteroaryl orR²¹ and R²² taken together form a carbonyl group;

wherein each alkyl, alkenyl, aryl, heterocyclyl or heteroaryl isoptionally substituted;

and geometric isomers or stereoisomers thereof and pharmaceutically,agriculturally or pesticidally acceptable salts thereof.

In preferred embodiments of formula II at least one of the followingapplies:

R¹ and R² are independently selected from hydrogen or —OH or takentogether form a carbonyl group;

R³ is hydrogen or —C₁-C₃ alkyl, especially hydrogen or methyl;

R⁴ and R⁵ are each hydrogen or R⁴ and R⁵ form a double bond or are —O—;

R⁶ is hydrogen or —C₁-C₃ alkyl, especially hydrogen;

R⁸ is hydrogen or —C₁-C₃ alkyl, especially hydrogen or methyl;

R¹⁰ is hydrogen;

R¹¹ is —OH, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl, —OC(O)C₁-C₂₀ alkyl,—OC(O)C₂-C₂₀ alkenyl, especially —OC(O)C₁-C₁₅ alkyl or —OC(O)C₂-C₁₅alkenyl, where each alkenyl group has one or more double bonds where thedouble bonds may be isolated or conjugated;

R¹² is —OH, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl, —OC(O)C₁-C₂₀ alkyl or—OC(O)C₂-C₂₀ alkenyl, especially -OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀alkenyl;

R¹³ and R¹⁴ are each independently hydrogen or —C₁-C₃ alkyl, especiallyhydrogen or methyl, more especially where both R¹³ and R¹⁴ are methyl;

R¹⁸ is —OH, —OC₁-C₆ alkyl, —OC₂-C₆ alkenyl, —OC(O)C₁-C₆ alkyl or—OC(O)C₂-C₆ alkenyl, especially —OH or —OC(O)C₁-C₃ alkyl;

R¹⁹ and R²⁰ are independently selected from hydrogen, —OH, —OC₁-C₃alkyl, —OC₂-C₃ alkenyl or R¹⁹ and R²⁰ taken together form a carbonylgroup, especially hydrogen, hydroxy or together a carbonyl group;

R²¹ is hydrogen;

R²² is —OH, —OC₁-C₆ alkyl, —OC₂-C₆ alkenyl, —OC(O)C₁-C₆ alkyl or—OC(O)C₂-C₆ alkenyl, especially —OH or —OC(O)C₁-C₃ alkyl;

R²³ is —OH or —OC₁-C₃ alkyl, especially —OH;

R²⁴ is —OH or —OC₁-C₃ alkyl, especially —OH.

In one particular embodiment, the compound is a compound of formula IIis12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-46):

In another embodiment the compound is12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-tigliaen-3-one(EBI-47):

In yet another particular embodiment, the compound is12-(dodeca-2,4,6-trienoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-59):

In still yet another particular embodiment, the compound is12-(deca-2,4-dienoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-61):

In yet another embodiment, the compounds is12,13-di-(2-methylbutanoyl)-1,2-2H-1,2,6,7-diepoxy-6-carboxy-4,5,9,12,13-pentahydroxy-tigliaen-3-one:

In yet another embodiment, the compound is12,13-di-(2-methylbutanoyl)-5,20-di-acetoyl-4,5,9,12,13,20-hexahydroxy-tigliaen-3-one:

The term “alkyl” refers to optionally substituted linear and branchedhydrocarbon groups having 1 to 20 carbon atoms. Where appropriate, thealkyl group may have a specified number of carbon atoms, for example,—C₁-C₆ alkyl which includes alkyl groups having 1, 2, 3, 4, 5 or 6carbon atoms in linear or branched arrangements. Non-limiting examplesof alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- andt-butyl, pentyl, 2-methylbutyl, 3-methylbutyl, hexyl, 2-methylpentyl,3-methylpentyl, 4-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, heptyl,octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl.

The term “alkenyl” refers to optionally substituted, unsaturated linearor branched hydrocarbons, having 2 to 20 carbon atoms and having atleast one double bond. Where appropriate, the alkenyl group may have aspecified number of carbon atoms, for example, C₂-C₆ alkenyl whichincludes alkenyl groups having 2, 3, 4, 5 or 6 carbon atoms in linear orbranched arrangements. Non-limiting examples of alkenyl groups include,ethenyl, propenyl, isopropenyl, butenyl, s- and t-butenyl, pentenyl, hexenyl, hept-1,3 -diene, hex-1,3 -diene, non-1,3,5-triene and the like.

The term “alkynyl” refers to optionally substituted unsaturated linearor branched hydrocarbons, having 2 to 20 carbon atoms, having at leastone triple bond. Where appropriate, the alkynyl group may have aspecified number of carbon atoms, for example, C₂-C₆ alkynyl whichincludes alkynyl groups having 2, 3, 4, 5 or 6 carbon atoms in linear orbranched arrangements. Non-limiting examples include ethynyl, propynyl,butynyl, pentynyl and hexynyl.

The terms “cycloalkyl” and “carbocyclic” refer to optionally substitutedsaturated or unsaturated mono-cyclic, bicyclic or tricyclic hydrocarbongroups. Where appropriate, the cycloalkyl group may have a specifiednumber of carbon atoms, for example, C₃-C₆ cycloalkyl is a carbocyclicgroup having 3, 4, 5 or 6 carbon atoms. Non-limiting examples mayinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,cyclohexenyl, cyclohexadienyl and the like.

“Aryl” means a C₆-C₁₄ membered monocyclic, bicyclic or tricycliccarbocyclic ring system having up to 7 atoms in each ring, wherein atleast one ring is aromatic. Examples of aryl groups include, but are notlimited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl.The aryl may comprise 1-3 benzene rings. If two or more aromatic ringsare present, then the rings may be fused together, so that adjacentrings share a common bond.

“Heterocyclic” or “heterocyclyl” refers to a non-aromatic ring having 3to 8 atoms in the ring and of those atoms 1 to 4 are heteroatoms, saidring being isolated or fused to a second ring selected from 3- to7-membered alicyclic ring containing 0 to 4 heteroatoms, wherein saidheteroatoms are independently selected from O, N and S. Heterocyclicincludes partially and fully saturated heterocyclic groups. Heterocyclicsystems may be attached to another moiety via any number of carbon atomsor heteroatoms of the radical and may be both saturated and unsaturated,which includes all forms of carbohydrate moieties. Non-limiting examplesof heterocyclic include pyrrolidinyl, pyrrolinyl, pyranyl, piperidinyl,piperazinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl,pyrazolinyl, dithiolyl, oxathiolyl, dioxanyl, dioxinyl, oxazinyl,azepinyl, diazepinyl, thiazepinyl, oxepinyl and thiapinyl, imidazolinyl,thiomorpholinyl, and the like.

The term “heteroaryl” as used herein means a stable monocyclic orbicyclic ring of up to 7 atoms in each ring, wherein at least one ringis aromatic and at least one ring contains from 1-4 heteroatoms,selected from sulfur, oxygen and nitrogen. Heteroaryl includes, but isnot limited to, oxazolyl, thiazolyl, thienyl, furyl, 1-isobenzofuranyl,pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isooxazolyl, pyridyl,pyrazinyl, pyrimidinyl, pyradazinyl, indolizinyl, isoindolyl, indolyl,purinyl, phthalazinyl, 1,2,3 -triazolyl, 1,2,4-triazolyl,1,2,3-oxadiazoyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,3,4-oxatriazolyl, 1,2,3,5-oxatriazolyl,1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3 -triazinyl, benzofuranyl,isobenzofuranyl, thionaphthenyl, isothionaphthenyl, indoleninyl,2-isobenzazolyl, 1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, isoindazolyl,indoxazinyl, benzoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl,quinazolinyl, naphthyridinyl, pyrido[3,4-b]pyridinyl,pyrido[3,2-b]pyridinyl, pyrido[4,3-b]pyridinyl, acridinyl, carbazolyl,quinaoxalinyl, pyrazolyl, benzotriazolyl, thiophenyl, isoquinolinyl,pyridinyl, tetrahydroquinolinyl, benzazepinyl, benzodioxanyl,benzoxepinyl, benzodiazepinyl, benzothiazepinyl and benzothiepinyl andthe like.

Each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl andheteroaryl group are optionally substituted with one or more substituentindependently selected from —F, —Cl, —Br, —I, —CN, —CF₃, —CO₂R²⁸,—COR²⁸, —N(R²⁸)₂, —NO₂, —NR²⁸OR²⁸, —ON(R²⁸)₂, —SOR²⁸, —SO₂R²⁸, —SO₃R²⁸,—SON(R²⁸)₂, —SON(R²⁸)₂, —SO₃N(R²⁸)₂, —P(R²⁸)₃, —P(═O)(R²⁸)₃, —OSi(R²⁸)₃,—OB(R²⁸)₂ wherein R²⁸ is as defined above.

Yet another aspect of the invention provides a pharmaceutically,agriculturally or pesticidally acceptable salt of a compound of formula(I) or formula (II).

The terms “pharmaceutically acceptable salts”, “agriculturallyacceptable salts” and “pesticidally acceptable salts” as used hereinrefer to salts which are toxicologically safe for systemic or topicaladministration to a human or animal or those that may be safely appliedto a plant or environment. The pharmaceutically, agriculturally orpesticidally acceptable salts may be selected from the group including,but not limited to, alkali and alkali earth, ammonium, aluminium, iron,amine, glucosamine, chloride, sulphate, sulphonate, bisulphate, nitrate,citrate, tartrate, bitarate, phosphate, carbonate, bicarbonate, malate,maleate, napsylate, fumarate, succinate, acetate, benzoate,terephthalate, pamoate, pectinate and s-methyl methionine salts,piperazine and the like.

It will also be recognised that compounds of the invention may possessasymmetric centres and are therefore capable of existing in more thanone stereoisomeric form. The invention thus also relates to compounds insubstantially pure isomeric form at one or more asymmetric centres e.g.,greater than about 90% ee, such as about 95% or 97% ee or greater than99% ee, as well as mixtures, including racemic mixtures, thereof. Suchisomers may be obtained by isolation from natural sources, by asymmetricsynthesis, for example using chiral intermediates, or by chiralresolution. The compounds of the invention may exist as geometricalisomers. The invention also relates to compounds in substantially purecis (Z) or trans (E) forms or mixtures thereof.

The compounds of the present invention may be obtained by isolation froma plant or plant part, or by derivatisation of the isolated compound, orby derivatisation of a related compound.

Yet another aspect of the invention provides a method of isolating oneor more compounds of formula (I) to formula (IV), which method includesthe step of extracting said one or more compounds from a plant or plantpart.

Preferably, the plant is of the genus Fontainea or Hylandia.

Preferably the species is Fontainea pancheri, Fontainea australis,Fontainea borealis, Fontainea fugax, Fontainea oraria, Fontaineapicrosperma, Fontainea rostrata, Fontainea subpapuana, Fontainea venosaor Hylandia dockrillii, especially Fontainea picrosperma, Fontaineavenosa or Hylandia dockrillii.

The parts of the plant may include fruit, seed, bark, leaf, flower,roots and wood.

Preferably the extract is obtained from the seed, bark and/or flowers.

For example, the biomass obtained from seeds, leaves, flowers and barkof the plant is subject to initial solvent extraction, for example witha polar solvent such as methanol. The initial extraction is thenconcentrated and diluted with water and subject to extraction with asecond solvent, for example, ethyl acetate. The solvent samples from thesecond extraction are pooled and subject to separation by preparativeHPLC fractionation. The fractions are analysed by analytical HPLC andpooled according to the retention time of compounds found in thesamples. The pooled fractions are weighed, bioassayed and analysed byanalytical HPLC. Further fractionation using one or more preparativeHPLC is performed to isolate specific compounds. Each compound isbioassayed and its structure identified by UV, NMR and massspectrometric techniques.

Other compounds of the invention may be obtained by derivatisingcompounds isolated from plants or parts of plants, especially from thegenus Fontainea, especially from the species Fontainea picrosperma,especially the seeds, bark and/or flowers of Fontainea picrosperma.

Derivatives of the natural compounds can be obtained by techniques knownin the art. For example, hydroxy groups may be oxidised, to ketones,aldehydes or carboxylic acids by exposure to oxidising agents such aschromic acid, Jones' reagent, KMnO₄, peracids such as mCPBA(metachloroperbenzoic acid) or dioxiranes such as dimethyldioxirane(DMDO) and methyl(trifluoromethyl)dioxirane (TFDO). Oxidising agents maybe chosen such that other functional groups in the molecule are or arenot also oxidised. For example, a primary alcohol may be selectivelyoxidised to an aldehyde or carboxylic acid in the presence of secondaryalcohols using reagents such as RuCl₂(PPh₃)₃-benzene. Secondary alcoholsmay be selectively oxidised to ketones in the presence of a primaryalcohol using Cl₂-pyridine or NaBrO₃-ceric-ammonium nitrate. Alcoholsmay be oxidised in the presence of double and triple bonds and withoutepimerisation at adjacent stereocentres using Jones' reagent with ourwithout Celite (or ammonium chloride). Alternatively, reagents chosenmay be less selective resulting in oxidation at more than one functionalgroup.

Hydroxy groups may also be derivatised by etherification or acylation.For example, ethers may be prepared by formation of an alkoxide ion inthe presence of base and reacting the alkoxide with an appropriatealkylhalide, alkenylhalide, alkynylhalide or arylhalide. Similarlyacylation may be achieved by formation of an alkoxide ion and reactionwith an appropriate carboxylic acid or activated carboxylic acid (suchas an anhydride).

Hydroxy groups may be derivatised to provide carbamates orthiocarbamates by reaction with isocyanates or isothiocyanates.

Carboxylic acids can be converted to thioesters or thioamides usingLawesson's reagent.

Acyl groups may be hydrolysed to provide alcohols by acid or basehydrolysis as known in the art and those alcohols can be derivatisedfurther as above.

Ketones may be reduced to secondary alcohols by reducing agents such aslithium aluminium hydride and other metal hydrides without reducingdouble bonds, including α-unsaturated ketones.

Double bonds and triple bonds may be reduced to single bonds usingcatalytic reduction, for example, H₂/Pd. Double bonds may also beoxidised to epoxides using oxidising agents such as per acids, forexample mCPBA or dioxiranes, such as DMDO and TFDO. Double bonds mayalso be subject to addition reactions to introduce substituents such ashalo groups, hydroxy or alkoxy groups and amines.

A person skilled in the art would be able to determine suitableconditions for obtaining derivatives of isolated compounds, for example,by reference to texts relating to synthetic methodology, examples ofwhich are Smith M. B. and March J., March's Advanced Organic Chemistry,Fifth Edition, John Wiley & Sons Inc., 2001 and Larock R. C.,Comprehensive Organic Transformations, VCH Publishers Ltd., 1989.Furthermore, selective manipulations of functional groups may requireprotection of other functional groups. Suitable protecting groups toprevent unwanted side reactions are provided in Green and Wuts,Protective Groups in Organic Synthesis, John Wiley & Sons Inc., 3^(rd)Edition, 1999.

Still yet another aspect of the invention provides a pharmaceuticalcomposition for treatment or prophylaxis of a disease or conditioncomprising an effective amount of one or more compounds of formula (I)or formula (II), or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier, diluent and/or excipient.

Dosage form and rates for pharmaceutical use and compositions arereadily determinable by a person of skill in the art.

Dosage forms include tablets, dispersions, suspensions, injections,solutions, syrups, troches, capsules, suppositories, aerosols,transdermal patches, creams, gels and the like. These dosage forms mayalso include injecting or implanting devices designed specifically for,or modified to, controlled release of the pharmaceutical composition.Controlled release of the therapeutic agent may be effected by coatingthe same, for example, with hydrophobic polymers including acrylicresins, waxes, higher aliphatic alcohols, polyactic and polyglycolicacids and certain cellulose derivates such as hydroxypropylmethylcellulose. In addition, the controlled release may be affected by usingother polymer matrices, liposomes and/or microspheres.

Pharmaceutically acceptable carriers and acceptable carriers forsystemic administration may also be incorporated into the compositionsof this invention.

Suitably, the pharmaceutical composition comprises a pharmaceuticallyacceptable excipient or an acceptable excipient. By “pharmaceuticallyacceptable excipient” is meant a solid or liquid filler, diluent orencapsulating substance that may be safely used. Depending upon theparticular route of administration, a variety of carriers, well known inthe art may be used. These carriers or excipients may be selected from agroup including sugars, starches, cellulose and its derivates, malt,gelatine, talc, calcium sulphate, vegetable oils, synthetic oils,polyols, alginic acid, phosphate buffered solutions, emulsifiers,isotonic saline, and pyrogen-free water.

Any suitable route of administration may be employed for providing ahuman or non-human patient with the pharmaceutical composition of theinvention. For example, oral, rectal, parenteral, sublingual, buccal,intravenous, intraarticular, intra-muscular, intra-dermal, subcutaneous,inhalational, intraocular, intraperitoneal, intracerebroventricular,transdermal and the like may be employed.

Pharmaceutical compositions of the present invention suitable foradministration may be presented in discrete units such as vials,capsules, sachets or tablets each containing a predetermined amount ofone or more pharmaceutically active compounds of the invention, as apowder or granules or as a solution or a suspension in an aqueousliquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oilemulsion or as a solution or suspension in a cream or gel. Suchcompositions may be prepared by any of the method of pharmacy but allmethods include the step of bringing into association one or morepharmaceutically active compounds of the invention with the carrierwhich constitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theagents of the invention with liquid carriers or finely divided solidcarriers or both, and then, if necessary, shaping the product in to thedesired presentation.

In powders, the carrier is a finely divided solid which is in a mixturewith the finely divided active component.

In tablets, the active component is mixed with the carrier having thenecessary binding capacity in suitable proportions and compacted in theshape and size desired.

The powders and tablets preferably contain from five or ten to aboutseventy percent of the active compound. Suitable carriers are magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as carrier providing acapsule in which the active component, with or without carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid formssuitable for oral administration.

For preparing suppositories, a low melting wax, such as admixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogenous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water-propylene glycol solutions. For example,parenteral injection liquid preparations can be formulated as solutionsin aqueous polyethylene glycol solution.

The compounds according to the present invention may thus be formulatedfor parenteral administration (e.g. by injection, for example bolusinjection or continuous infusion) and may be presented in unit dose formin ampoules, pre-filled syringes, small volume infusion or in multi-dosecontainers with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilising and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilisation from solution, for constitution witha suitable vehicle, e.g. sterile, pyrogen-free water, before use.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavours,stabilizing and thickening agents, as desired.

Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, or other well known suspending agents.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavours, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

For topical administration to the epidermis the compounds according tothe invention may be formulated as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase and will in general also contain one or more emulsifying agents,stabilising agents, dispersing agents, suspending agents, thickeningagents, or colouring agents.

Formulations suitable for topical administration in the mouth includelozenges comprising active agent in a flavoured base, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert base such as gelatin and glycerin or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier.

Solutions or suspensions are applied directly to the nasal cavity byconventional means, for example with a dropper, pipette or spray. Theformulations may be provided in single or multidose form. In the lattercase of a dropper or pipette, this may be achieved by the patientadministering an appropriate, predetermined volume of the solution orsuspension. In the case of a spray, this may be achieved for example bymeans of a metering atomising spray pump. To improve nasal delivery andretention the compounds according to the invention may be encapsulatedwith cyclodextrins, or formulated with their agents expected to enhancedelivery and retention in the nasal mucosa.

Administration to the respiratory tract may also be achieved by means ofan aerosol formulation in which the active ingredient is provided in apressurised pack with a suitable propellant such as a chlorofluorocarbon(CFC) for example, dichlorodifluoromethane, trichlorofluoromethane, ordichlorotetrafluoroethane, carbon dioxide, or other suitable gas. Theaerosol may conveniently also contain a surfactant such as lecithin. Thedose of drug may be controlled by provision of a metered valve.

Alternatively the active ingredients may be provided in the form of adry powder, for example a powder mix of the compound in a suitablepowder base such as lactose, starch, starch derivatives such ashydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).

Conveniently the powder carrier will form a gel in the nasal cavity. Thepowder composition may be presented in unit dose form for example incapsules or cartridges of, e.g., gelatin, or blister packs from whichthe powder may be administered by means of an inhaler.

In formulations intended for administration to the respiratory tract,including intranasal formulations, the compound will generally have asmall particle size for example of the order of 1 to 10 microns or less.Such a particle size may be obtained by means known in the art, forexample by micronization.

The active compounds of formula (I) and formula (II) and of thecompositions of this invention are present in an amount sufficient toprevent, inhibit or ameliorate a disease or condition. Suitable dosagesof the compounds of formula (I) or formula (II) and the pharmaceuticalcompositions containing such may be readily determined by those skilledin the art.

In a still further aspect of the invention, there is provided a methodof treating or prophylaxis of a disease or condition comprisingadministering to a subject in need of such treatment an effective amountof one or more compounds according to formula (I) or formula (II), or apharmaceutically acceptable salt thereof.

In yet another aspect of the invention, there is provided the use of oneor more of the compounds according to formula (I) or formula (II), or apharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment or prophylaxis of a disease or condition.

Non-limiting examples of a diseases or a condition are a bacterialinfection, a parasitic infestation and a cell proliferative disorder. Innon-limiting embodiments compounds of formula (I) or formula (II) haveone or more activities selected from antiparasitic activity (e.g.against an endoparasite and/or an ectoparasite, such as, Haemonchuscontortus), antibiotic activity (e.g. against Bacillus subtilis),antiprotozoal activity (e.g. against Giardia sp. Portland) cytotoxicactivity (e.g. against a basal cell carcinoma and/or a squamous cellcarcinoma and/or a melanoma and/or antitumour activity (e.g. against aleukemia, a melanoma, a prostate cancer, a breast cancer, an ovariancancer and/or other solid tumour cancers).

In one aspect of the invention, there is provided a method of treatingor preventing a bacterial infection in a subject comprisingadministering to a subject a compound of formula (I) or formula (II) ora pharmaceutically acceptable salt thereof.

In preferred embodiments, the compound of formula (I) and formula (II)is12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-46).

The bacterial infection may be caused by a Gram positive or Gramnegative bacteria, especially a Gram positive bacteria. Non-limitingexamples of bacteria that are controlled by the compounds of theinvention include bacteria of the Genus Bacillus, such as B. subtilis,B. anthracis, B. cereus, B. fermis, B. licheniformis, B. megaterium, B.pumilus, B. coagulans, B. pantothenticus, B. alvei, B. brevis, B.circubins, B. laterosporus, B. macerans, B. polymyxa, B.stearothermophilus, B. thuringiensis and B. sphaericus; Staphlococcussuch as S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus;Streptococcus, for example, S. pyrogenes, S. pneumoniae, S. alagactiae,S. dysgalactiae, S. equisimilis, S. equi, S. zooepidemicus, S.anginosus, S. salwarius, S. milleri, S. sanguis, S. mitior, S. mutans,S. faecalis, S. faecium, S. bovis, S. equinus, S. uberus and S. avium;Aerococcus spp., Gemella spp., Corynebacterium spp., Listeria spp.,Kurthia spp., Lactobacillus spp., Erysipelothrix spp., Arachnia spp.,Actinomyces spp., Propionibacterium spp., Rothia spp., Bifidobacteriumspp., Clostridium spp., Eubacterium spp., Nocardia spp. andMycobacterium spp.

In another aspect of the invention there is provided a method oftreating or preventing a parasitic infection in a subject or plantcomprising administering to a subject a compound of formula (I) orformula (II) or a pharmaceutically acceptable salt thereof.

In preferred embodiments, the parasite is a helminth (worm), especiallynematodes, trematodes and cestodes, such as Haemonchus contortus,Trichinella spiralis, H. placei, Bursaphelenchus xylophilus, Ostertagiacircumcincta, O. ostertagi, Mecistocirrus digitatus, Trychostrongylusaxei, Trichuris trichiura, T. vulpis, T. campanula, T. suis, T. ovis,Bunostomum trigonocephalum, B. phleboyomum, Oesophagostomum columbianum,O. radiatum, Cooperia curticei, C. punctata, C. oncophora, C. pectinata,Strongyloides papillosus, Chabertia ovina, Ancylostoma duodenale, A.braziliense. A. tubaeforme, A. caninum, Ascaris lumbricoides, Enterobiusvermicularis, E. gregorii, Ascaris lumbricoides, Paragonimus Westermani,Clonorchis sinensis, Fasciola hepatica, Taenia solium, T saginata,Capillaria aerophila, Necator americanus, species of the genusTrichuris, Baylisascaris, Aphelenchoides, Meliodogyne, Heterodera,Globodera, Nacobbus, Pratylenchus, Ditylenchus, Xiphinema, Longidorus,Trichodorus, Nematodirus.

In this embodiment, preferred compounds include12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (EBI-46),12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-47) and12-(deca-2,4-dienoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-61).

In yet another aspect of the invention, there is provided a method oftreating or preventing a cell proliferative disorder in a subjectcomprising administering to a subject a compound of formula (I) orformula (II) or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the cell proliferative disorder is a cancer,especially where the cancer is selected from leukaemia, melanoma,prostate cancer, breast cancer, ovarian cancer, basal cell carcinoma,squamous cell carcinoma, fibrosarcoma, colon cancer, lung cancer,neoplasms and other solid tumour cancers. In one embodiment, the cellproliferative disorder is a dermatological neoplasm, particularly equinesarcoides.

In other embodiments, the cell proliferative disorder is a non-cancerousproliferative disorder. Examples of such non-cancerous proliferativedisorders include dermatological disorders such as warts, keloids,psoriasis, proud flesh disorder and also reduction in scar tissue andcosmetic remodelling.

In preferred embodiments of the treatment or prevention ofdermatological cell proliferative disorders, the therapy is preferablytopical or administered intra-lesionally to provide a localised effect.

Without wishing to be bound by theory, the compounds of the inventionare thought to bind to the C₁ region of PKC activating signallingpathways such as the MAP kinase pathway resulting in release ofcytokines (Challacombe et al., J. Immunol., 2006, 177:8123-32; Cozzi etal., Cancer Res., 2006, 66:10083-91). This localised stimulation ofinflammatory response prevents, reduces or removes unwanted cellsthereby treating or preventing cell proliferative disorders.Accordingly, in another aspect of the invention, there is provided amethod of stimulating a localised inflammatory response comprisingadministering to a subject a compound of formula (I) or formula (II) ora pharmaceutically acceptable salt.

If the compound of the invention is administered locally to treat a cellproliferative disorder, it may be administered at a higher dosage levelthan if administered systemically. For systemic administration to treatthe subset of tumours and lymphoid neoplasms that are highly sensitiveto growth inhibition in culture, a lower dose of compound isadministered, for example, in the μg/kg dosage. Whereas, localisedadministration may be up to 1000 times greater, for example in the mg/kglevel. For the drug class of PKC activators the mechanism of action atlow systemic doses is likely to involve direct action on the tumourcells producing senescence (Cozzi et al., 2006) or apoptosis (Hampson etal., Blood, 2005, 106:1362-8); whereas at high, localised doses the hostresponse, associated with a local inflammatory response, is alsoimportant (Challacombe et al., 2006)

In some instances, it is advantageous to administer the compounds of theinvention together with an anti-inflammatory agent to reduce unwantedinflammatory responses. Examples of suitable anti-inflammatory agentssuch as ibuprofen, aspirin, pentoxifylline, dexamethasone, prednisolone,prednisone, cortisone, beclamethasone, fluticasone, hydrocortisone,methyl-prednisolone, triamcinolone, budesonide, betamethasone, naproxen,nabumetone and Cox-2 inhibitors such as celecoxib, rofecoxib andvaldecoxib. The compounds of the invention may be administeredsimultaneously, separately or sequentially with the anti-inflammatoryagent.

In this embodiment, preferred compounds include12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-46),12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-47),12-(deca-2,4-dienoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-61) and12-(dodeca-2,4,6-trienoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-59).

In yet another embodiment of the present invention, there is provided amethod of treating or preventing a protozoan infection in a subjectcomprising administering to a subject a compound of formula (I) orformula (II) or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the protozoan infection is selected fromGiardia spp., Trichomonas spp., African trypanosomiasis, amoebicdysentery, babesiosis, balantidial dysentery, Chaga's disease,coccidiosis, malaria and toxoplasmosis, especially Giardia spp. andTrichomonas spp. infections.

In this embodiment, a preferred compound is12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (EBI-47).

In yet another aspect of the present invention, there is provided a useof a compound of formula (I) or formula (II) in the manufacture of amedicament for treating or preventing a bacterial infection, a parasiticinfection, a protozoan infection or a cell proliferative disorder.

The term “subject” as used herein includes humans, primates, livestockanimals (eg. sheep, pigs, cattle, horses, donkeys), laboratory testanimals (eg. mice, rabbits, rats, guinea pigs), companion animals (eg.dogs, cats), birds (eg. chickens, ducks, geese, parrots, cockatoos,pigeons, finches, raptors, ratites, quail, canaries), captive wildanimals (eg. foxes, kangaroos, deer) and reptiles (eg. lizards andsnakes). Preferably, the mammal is human, a livestock animal, acompanion animal or a laboratory test animal. Even more preferably, themammal is a human, a livestock animal or a companion animal.

An “effective amount” means an amount necessary at least partly toattain the desired response, or to delay the onset or inhibitprogression or halt altogether, the onset or progression of a particularcondition being treated. The amount varies depending upon the health andphysical condition of the individual to be treated, the taxonomic groupof individual to be treated, the degree of protection desired, theformulation of the composition, the assessment of the medical situation,and other relevant factors. It is expected that the amount will fall ina relatively broad range that can be determined through routine trials.An effective amount in relation to a human patient, for example, may liein the range of about 0.1 ng per kg of body weight to 1 g per kg of bodyweight per dosage. The dosage is preferably in the range of 1 μg to 1 gper kg of body weight per dosage, such as is in the range of 1 mg to 1 gper kg of body weight per dosage. In one embodiment, the dosage is inthe range of 1 mg to 500 mg per kg of body weight per dosage. In anotherembodiment, the dosage is in the range of 1 mg to 250 mg per kg of bodyweight per dosage. In yet another embodiment, the dosage is in the rangeof 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mgper kg of body weight per dosage. In yet another embodiment, the dosageis in the range of 1 μg to 1 mg per kg of body weight per dosage. Dosageregimes may be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily, weekly,monthly or other suitable time intervals, or the dose may beproportionally reduced as indicated by the exigencies of the situation.

Reference herein to “treatment” and “prophylaxis” is to be considered inits broadest context. The term “treatment” does not necessarily implythat a subject is treated until total recovery. Similarly, “prophylaxis”does not necessarily mean that the subject will not eventually contracta disease condition. Accordingly, treatment and prophylaxis includeamelioration of the symptoms of a particular condition or preventing orotherwise reducing the risk of developing a particular condition. Theterm “prophylaxis” may be considered as reducing the severity or onsetof a particular condition. “Treatment” may also reduce the severity ofan existing condition.

In another aspect of the invention, the compounds of the invention aresuitable for use as a pesticide. The invention therefore furtherprovides a pesticidal composition comprising a compound of formula (I)or formula (II) or an agriculturally or pesticidally acceptable saltthereof and a pesticidally acceptable carrier.

The pesticidal composition is preferably an insecticidal composition andmay be in the form of an emulsifiable concentrate, a flowable, awettable powder, a soluble powder, a solution, an aerosol, a dust, agranule or a bait. A person skilled in the formulation of pesticidalcompositions would be able to prepare such formulations.

Suitable carriers for pesticidal compositions include, but are notlimited to, oils, especially petroleum oils, emulsifiers, solvents suchas water or hydrocarbons, surfactants, aerosol spray components such asCFCs, talc or clay.

In yet another aspect of the invention, there is provided a method ofcontrolling infestations of pests in a subject or an environmentcomprising applying a pesticidally effective amount of a compound offormula (I) or formula (II) to a subject or an environment infested witha pest.

The agricultural pest is preferably an insect, especially flies,beetles, grasshoppers, locusts, butterflies and moths and their larvaeor nymphs, especially the flies (Diptera) such as true flies, fleas,ticks, lice, mosquitoes, gnats and midges.

In some embodiments, the pest infests plants. Examples of such pestsinclude, but are not limited to, Acyrthosiphon kondoi (blue-greenaphid), Acyrthosiphon pisum (pea aphid), Agrotis spp. (cutworm),Agrypnus variabilis (sugarcane wireworm), Anoplognathus spp. (christmasbeetles), Aphodius tasmaniae (blackheaded pasture cockchafer),Austroasca alfalfae (lucerne leaf hopper), Bathytricha truncate(sugarcane and maize stemborer), Bemisia tabaci (whitefly), Brachycaudushelichrysi (leaf curl plum aphid), Brevicoryne brassicae (cabbageaphid), Bruchophagus roddi (lucerne seed wasp), Bruchus pisorum (peaweevil), Bryobia spp. (bryobia mite), Ciampa arietaria (brown pasturelooper), Chortoicetes terminifera (Australian plague locust),Chrysodeitis angentifena (tobacco looper), Chrysodeitis eriosoma (greenlooper), Contarinia sorghicola (sorghum midge), Deroceras spp. (slugs),Diachrysia oricalcea (soybean looper), Etiella behrii (lucerne seed-webmoth), Frankliniella schultzei (tomato thrips), Graphognathus leucoloma(white fringed weevil), Halotydeus destructor (redlegged earth mite),Hednota pedionoma (pasture webworm), Helicoverpa armigera (cornearworm), Helicoverpa punctigera (native budworm), Helix spp. (snails),Heteronychus arator (African black beetle), Leucania convecta (commonarmyworm), Lipaphis erysimi (turnip aphid), Listroderes difficilis(vegetable weevil), Melanacanthus scutellaris (brown bean bug),Merophyas divulsana (lucerne leaf roller), Myzus persicae (green peachaphid), Nala lividipes (black field earwig), Mythimna convector (commonarmyworm), Nezara viridula (green vegetable bug), Nysius vinitor(rutherglen bug), Nysius clevelandensis (grey cluster bug), Oncoperarufobrunnea (underground grass grub), Orondina spp. (false wireworm),Othnonius batesi (black soil scarabs), Penthaleus major (blue oat mite),Persectania ewingii (southern armyworm), Petrobia lateens (brown wheatmite), Pieris rapae (cabbage white butterfly), Piezodorus hybneri(redbanded shield bug), Plutella xylostella (cabbage moth/diamondbackmoth), Rhopalosiphum maidis (corn aphid), Sericesthis spp. (smallbrownish cockchafers), Sitona discoideus (sitona weevil), Sminthurusviridis (lucerne flea), Spodoptera exigua (lesser armyworm), Spodopteraletura (cluster caterpillar Spodoptera mauritia (lawn armyworm),Stomopteryx simplexella (soybean moth), Tetranychus ludeni (bean spidermite), Tetranychus urticae (two spotted mite), Therioaphis trifolii f.maculata (spotted alfalfa aphid), Thrips tabaci (onion thrips), Thripsimaginis (plague thrips), Zizina labradus (grass blue butterfly),Zygrita diva (lucerne crown borer).

In other embodiments, the pests infest subjects and/or environmentsother than plants. Examples of such pests include, but are not limitedto, lice, ants including Camponotus spp., Lasius alienus, Acanthomyopsinterjectus, Monomorium pharaonis, Solenopsis molesta, Tetramoriumcaepitum, Monomorium minimum, Prenolepis impairs, Formica exsectoides,Iridomyrmex pruinosus, Cremastogaster lineolata, Tapinoma sessile,Paratrechina longicornis, cockroachs, mosquitos, bed bugs includingLeptoglassus occidentalis, Acrosternum hiare, Chlorochroa sayi, Podiusmaculiventris, Murgantia histrionica, Oncopeltus fasciatus, Nabisalternatus, Leptopterna dolabrata, Lygus lineolaris, Adelpocorisrapidus, Poecilocapsus lineatus, Orius insidiosus, Corythucha ciliata,bees, wasps, black widow spider, booklice, boxelder bug, brown reclusespider, clothes moths including Tineola spp., Tinea spp., Trichophagaspp., carpet beetles, centipedes, clover mites, cluster and face flies,cigarette and drugstore beetles, crickets including Acheta spp., Gryllusspp., Gryllus spp., Nemobius spp., Oecanthus spp., Ceuthophilus spp.,Neocurtilla spp., daddy-long-legs, domestic flies, drain flies, earwigs,European hornet, fleas including Ctenocephalides Fels, Ctenocephalidescanis, Ctenocephalides spp., Nosopsyllus fasciatus, Nosopsyllus spp.,Xenopsylla cheopis, Xenopsylla spp., Cediopsylla simplex, Cediopsyllaspp., fungus gnats, ground beetles, hide and larder beetles,horse/cattle/deer/pig flies, house dust mites including Dermatophagoidesfarinae, Dermatophagoides pteronyssinus, Dermatophagoides spp., mitesincluding Ornithonyssus sylviarum, Dermanyssus gallinae, Ornithonyssusbacoti , Liponyssoides sanuineus , Demodex folliculorum, Sarcoptesscabiei hominis, Pyemotes tritici , Acarus siro, Tyrophagusputrescentiae, Dermatophagoides sp., human lice, humbacked flies, Indianmeal moth, millipedes, mud daubers, multicolored asian lady beetle,house borer, midges and crane flies, periodical and “dog-day” cicadas,powderpost beetles, roundheaded and flatheaded borers, pseudoscorpions,psyllids or jumping plant lice, spider beetles, sac spiders, sapbeetles, termites, silverfish and firebrats, sowbugs and pillbugs,springtails, stinging hair caterpillars, tarantulas, vinegar flies,wasps and hornets, wharf borer, woods cockroach, yellowjacket wasps,fungus beetles, seed weevils, sawtoothed and merchant grain beetles,confused and red flour beetles, granery and rice weevils, indi an mealmoth, mealworms, drain flies, ticks including Dermacentar spp., Ixodesspp., Rhipicenphalus spp. , carpenter bees, fleas, assassin bugs, humanlice, chiggers, mystery bugs, european hornet, stinging haircaterpillars, black-legged tick, mayflies, black flies, horsehair worms,crickets, gypsy moths, grasshoppers, gnats, midges, locusts, mosquitoesincluding Aedes albopictus, Aedes Canadensis Aedes triseriatus, Aedestivittatus, Aedes vexans, Aedes spp., Anopheles quadrimaculatus,Anopheles spp., Coquillettidia perturbans, Coquillettidia spp., Culexpipiens, Culex spp.

An agriculturally effective amount may be determined by those skilled inthe art using known methods and would typically range from 5 g to 500 gper hectare.

The compounds of the invention may be applied to any environment inwhich pests are present. For example, an environment in whichagriculture is carried out, for example, the growing of crops, trees,and other plants of commercial importance. The agricultural environmentincludes not only the plant itself, but also the soil and area aroundthe plants as they grow and also areas where parts of plants, forexample, seeds, grains, leaves or fruit, may be stored. The environmentmay also be a household environment or industrial environment.

A “household environment” includes environments that are inhabited byhumans or animals and may include indoor environments such as carpets,curtains, cupboards, bedding and the air inside a house. An “industrialenvironment” includes environments which are used for industrialpurposes such as manufacture, storage or vending of products. Industrialenvironments include warehouses, manufacturing plants, shops, storagefacilities and the like.

In this aspect, preferred compounds of formula (I) and formula (II)include12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-47),12-(deca-2,4-dienoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-61) and12-(dodeca-2,4,6-trienoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-59).

The invention further provides use of a compound of formula (I) orformula (II) as an pesticide.

Accordingly, the compound of formula (I) or formula (II) may beformulated in an appropriate manner for delivery to subjects, crops,pastures, forests and other agricultural environments, or household orindustrial environments, preferably for the alleviation and/oreradication of one or more insect pests.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Flowchart for initial solvent extraction of compounds of formula(I);

FIG. 2A: Flowchart showing the solvent partition for the aqueousconcentrate obtained from the extraction shown in FIG. 1;

FIG. 2B: Flowchart showing the solvent partition for the ethyl acetateresidue obtained from the extraction shown FIG. 1;

FIG. 3: Flowchart showing the steps in preparative HPLC chromatography;

FIG. 4: Graphically represents the selective inhibition of cell growthin culture by EBI-46;

FIG. 5: Graphically represents the results of topical treatment of B16tumours in C57/B6 mice with EBI-46 (once a day application for threedays starting from day 5);

FIG. 6: Graphically represents the results of topical treatment of LK2mouse SCC in nude mice;

FIG. 7: Graphically represents the inhibition of growth of LKC SSCtumours by topical application of EBI-46; and

FIG. 8: Graphically represents the effect of injected EBI-46 on LK-2 SCCtumours.

DETAILED DESCRIPTION

Activity Screening

A solvent extraction sample from Fontainea picrosperma containingcompounds of formula (I) and formula (II) were tested to determinetherapeutical activity by screening in (A) a range of MicrobialScreening Technologies (MST) bioassays, notably NemaTOX, ProTOX,MycoTOX, and CyTOX and DipteraTOX, and (B) a range of anticancer assays.For ease of description these bioassays will be described briefly priorto the extraction and chemical structure elucidation methodologies.

MST Bioassays Included:

NemaTOX (alternatively referred to herein as Ne) is an anthelminticbioassay, applicable to all parasitic nematodes with free-living lifecycle stages, and can be used as a screen to detect activity and definethe species spectrum of compounds against parasitic nematodes andexamine the impact of pre-existing resistance to other anthelminticclasses on potency. Haemonchus contortus was utilised for this assay.

The effect on larval development was determined in this assay by themethod described by Gill et al. (1995) Int. J. Parasitol. 25: 463-470.Briefly, in this assay nematode eggs were applied to the surface of anagar matrix containing the test sample and allowed to develop through tothe L3, infective stage (6 days). At this time the stage of larvaldevelopment reached and any unusual features (deformity, paralysis,toxicity) were noted by microscopic examination.

ProTOX, (alternatively referred to herein as Bs) is an antibacterialbioassay, broadly applicable to most aerobic and anaerobic bacteria. Thebioassay features a solid phase agar base into which the test compoundhas been incorporated together with a chromogen. As the bacteriamultiply in the well, the chromogen is metabolised from blue in atwo-step process to a colourless compound. Compounds with potentbactericidal activity inhibit bacterial metabolism of the chromogenwhile bacteriostatic compounds induce limited metabolism as indicated byan intermediate pink colour. ProTOX is broadly applicable to a range ofgram-positive and gram-negative bacteria under aerobic andmicroaerophilic conditions. ProTOX assays were carried out usingBacillus subtilis.

Briefly, in ProTOX, the bacteria (24 hour broth) were applied to thesurface of an agar matrix containing the test sample and allowed to growfor 48 hours. The assay was monitored at 24 and 48 hours and the activewells noted. Known antibiotics yield consistent colour transitions whichare concentration and time dependent. These patterns provide animportant guide to the early recognition of interesting characteristics.Bactericidal actives were assessed as having no colour change at both 24and 48 hours while bacteriostatic actives were assessed as active at 24hours but less potent or inactive at 48 hours.

MycoTOX (alternatively referred to herein as Tr) is a non-chromogenicbioassay used to detect activity against filamentous fungal pathogens ofplants and animals. The bioassay features a solid phase agar base intowhich the test compound has been incorporated. As the growth patterns offilamentous fungi are readily apparent on the agar surface the extent ofmycelial growth, sporulation (if relevant to the species underinvestigation) and colour changes with maturation are measured.Compounds with potent antifungal activity inhibit germination of fungalspores and provide a stark contrast to wells containing inactivecompounds with the excessive fungal growth. Lower concentrations of suchcompounds, or compounds exhibiting a more fungistatic mode of action,show reductions in mycelial growth, extent of sporulation or reductionsin other characteristic patterns of colony maturation.

MycoTOX, involves a fungus (spore suspension or mycelial fragments)being applied to the surface of an agar matrix containing the testchemical and allowed to grow for a period of up to a week (depending onspecies). The assay is monitored at two discrete times to identify keydevelopment phases in the life cycle (for example mycelial growth andextent of sporulation) and the active wells noted. The monitoring timesare dependent on the fungal species under investigation.

The MycoTOX assays were carried out using Trichophyton rubrum. TheMycoTOX test is alternatively referred to as Tr to indicate the use ofTrichophyton rubrum.

CyTOX (alternatively referred to herein as Cy) is a microtitre platebioassay use to identify potential antitumour actives. CyTOX is achromogenic bioassay with broad application to a wide range of tumourand non-tumour cell lines. The colour transitions in CyTOX areproportional to cell metabolism and turnover and hence offer usefulrecognition patterns to support the diagnostic classification of activeswithin a framework of known cytotoxic and antitumour actives.

CyTOX features a liquid media into which the test compound has beenincorporated together with a novel chromogen. As the cells grow anddivide the chromogen is metabolised from purple in a single step processto a colourless metabolite. CyTOX was undertaken using NS1 murinemyeloma cell line as a guide to mammalian cell toxicity.

Briefly, in CyTOX the cells were applied to the media containing thetest chemical and allowed to grow for 72 hours. The assay was monitoredat 24, 48 and 72 hours and the active wells identified.

DipteraTOX,

DipteraTOX is referred to herein as DipG, DipP and DipH. DipG representsno grazing of larva. DipP represents no pupae formation and Dip Hrepresents no hatching of flies. A value of A in DipG, Dip P or Dip Hrepresents very active and a value of P represents active. In DipteraToxthe fly eggs were applied to the surface of an agar matrix containing250 μg per mL of the test chemical and allowed to hatch, develop andpupate for a period up of two weeks. The assay was monitored at twodiscrete times to determine the extent of grazing of the agar matrix atWeek 1 and the presence of adult flies at Week 2. Activity was scoredqualitatively as active or inactive at Days 7 and 14 to denote failureto feed and failure to development to the adult stage, respectively.

TriTOX (Alternatively Referred to Herein as Gi)

is a microtitre plate based chromogenic bioassay for the screening ofanti-protozoan activity of pathogenic, anaerobic/microaerophilicprotozoans for example Giardia spp. and Trichomonas spp. The bioassaysare run under anaerobic conditions and features species specificchromogens. The minimum inhibitory concentrations (approximate LD99) aredetermined by the following method: stock solutions of the unknowns areserially diluted ½ to give 12 concentrations over a 2,048-fold range.Aliquots of each concentration(s) are applied to the wells of 96-wellmicrotitre plates and diluted with media. Test substances are scored asactive or inactive based on the chromogen colour change. The lowestconcentration at which the compound is active is noted as the minimuminhibitory concentration (MIC). Additionally, microscopic inspection iscarried out to identify any patterns of morphological change that may beconsistent with a type of toxicity and therefore mode of action. Giardiaspp. was utilized for this assay.

Anticancer Assays Included:

SRB Assay for Inhibition of Growth of Cells Cultured as Monolayers

Briefly, a range of tumour cells including mouse melanoma, humanmelanoma, mouse squamous cell carcinoma, human breast, human colon,human leukemia and human lung and normal human cells including normalhuman fibroblasts were seeded at 2-5,000 per microtitre well (96-wellplate) in 10% FCS-RPMI 1640 culture medium, treated, and allowed to growuntil the controls were nearly confluent (5-6 days). The wells were thenwashed twice with PBS, fix with ethanol for a minimum of 5 minutes andwashed with water. SRB solution (50 μL of 0.4% in 1% acetic acid) wasadded and left at room temperature for a minimum of 15 minutes. Theplate was washed rapidly with tap water and then twice with 1% aceticacid. After addition of 100 μL/well of 10 mM Tris base (unbuffered,pH>9), plates were left for a minimum of 5 minutes, then the absorbancewas read at 564 nm on the ELISA reader, with a 3 second prior shaking.After subtraction of a blank (wells with no cells, absorbance typicallyabout 0.04), growth inhibition was calculated as % of the untreatedcontrol and plotted against dose.

QIMR Assay for Inhibition of Growth of Cells Cultured in Suspension

Briefly, a suspension of a range of tumour cells including mousemelanoma, human melanoma, mouse squamous cell carcinoma, human breast,human colon, human leukemia and human lung and normal human cellsincluding normal human fibroblasts were seeded into round-bottommicrotitre plates in 10% FCS-RPMI 1640 culture medium, treated andallowed to grow for 5-6 days. To measure cell growth, 20 μL of acombined MTS/PMS solution (Promega Cell ProliferationAssay Kit Cat#G5430) was added to each well of the 96 well assay plate. After 1-4hours the plates were placed in the ELISA plate reader and theabsorbance read at 490 nm. After subtraction of a blank (wells with nocells, absorbance typically about 0.4), growth inhibition was calculatedas % of the untreated control and plotted against dose.

Topical Treatment of Tumours on Mice

Tumour cells were injected subcutaneously (2 million cells/site in 50 μLculture medium for LK2 cells, 0.5 million for B16) into 4 sites each onthe flanks of nude mice (for LK2 cells) or C57BL/6 mice (B16 mousemelanoma cells). When tumours became visible (2-4 mm diameter) five to10 days after injection of cells, 37 μg of EBI-46 in 25 μL, ofisopropanol gel was applied to each tumour site, each day for 3 days.Tumour size was measured with calipers at appropriate intervals.

Intralesional Injection at the Tumour Site on Mice

Squamous Cell Carcinoma (SCC) tumours were established by the samemethod as for topical treatment but were allowed to grow for 15 daysbefore drug treatment. 7 μg EBI-46 in 20 μL, saline was injected intoeach of 3 sides of the tumour. Tumour size was measured with calipers atappropriate intervals.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

EXAMPLE 1

Methods

Extraction

Biomass samples, including seeds, leaves and bark, from Fontaineapierosperma where collected and subject to the following extractionprocess. These samples and their subsequent fractions are referred inthe below example as EB548.

Phase 1—Extraction

The biomass is generously covered with methanol and shaken (˜2 L,overnight) followed by filtration to give the first extract. Thisprocess is repeated a second time (˜2 L, ˜5 hours) to generate thesecond extract. Each extract is examined by analytical HPLC andbioassayed (FIG. 1). The sequential methanol extracts are combined andthe solvent removed by rotary evaporation to afford an aqueousconcentrate.

Phase 2—Solvent Partition

The aqueous concentrate from the extraction is diluted with water to 400mL. The diluted sample (code ‘Cr’) is subsampled for HPLC and bioassay,then shaken with an equal volume of ethyl acetate (EtOAc) in aseparatory funnel and the individual layers, EtOAcl and H2O1, collected.Note, occasionally a precipitate would form that was insoluble in eitherlayer. This precipitate was collected by filtration and dissolved inmethanol (code ‘Me’). The lower aqueous layer (H2O1) was twice moreextracted with ethyl acetate to give EtOAc2 and EtOAc3 along with theremaining H2O3 layer. Subsamples of all layers are examined byanalytical HPLC and bioassay (FIG. 2A).

The sequential ethyl acetate extracts are pooled and the solvent removedby rotary evaporation to afford a residue that is weighed. On occasions,analytical HPLC indicated the EtOAc extract contained considerableamounts of extremely lipophilic (RT>9 minutes) material. To remove thismaterial a 10:9:1-hexane:methanol:water partition was performed (FIG.2B).

Phase 3—Preparative HPLC Fractionation

The residue from the solvent partition is investigated by analyticalHPLC to find optimum chromatographic conditions for separation of themetabolites present. Using these optimum conditions the residue (˜2 g)is fractionated by preparative reverse phase HPLC (C18, singleinjection) into 100 fractions (FIG. 3). Subsamples of all 100 fractionsare examined by analytical HPLC. After analysis of the HPLC traces, the100 fractions are consolidated into 20 to 30 pooled fractions (pools),some of which may be >80% pure. These pooled fractions are weighed,bioassayed and examined by analytical HPLC.

Solvent Partition Summary for EB548

Biomass samples of Fontainea picrosperma under went extraction andsolvent partitioning, using phase 1 and 2 described above. Table 1summarises the amounts of extractable material obtained after solventpartitioning with ethyl acetate.

TABLE 1 Weights after Ethyl Acetate Partition of Extracts Sample Weight¹EtOAc² % Ext.³ HPLC Comment EB548 318 68.4 21.5% Excellent ¹Weight:Total sample weight in grams of plant material supplied and used for thestudy. ²EtOAc: Ethyl acetate extractables. ³% Ext.: Ethyl acetateextractables expressed as a percentage of the total sample weight.Preparative HPLC

The preparative HPLC was carried out on a system consisting of twoShimadzu LC-8A Preparative Liquid Chromatographs with static mixer,Shimadzu SPD-M10AVP Diode Array Detector and Shimadzu SCL-10AVP SystemController. The column used was 50×100 mm (diameter×length) packed withC18 Platinum EPS (Alltech).

Approximately 2 grams of ethyl acetate extracted material was dissolvedin dimethyl sulphoxide (4 mL) and subjected to preparative HPLC withtypically conditions being 60 mL/min with gradient elution of 30% to100% acetonitrile/water over 20 minutes followed by acetonitrile for 10minutes. One hundred fractions (20 mL) were collected, evaporated undernitrogen, and then combined on the basis of HPLC analysis.

UV Analysis

UV spectra were acquired during HPLC with the Shimadzu SPD-M10AVP DiodeArray Detector as mentioned above.

NMR Analysis

All NMR spectra were acquired in d6-dimethyl sulphoxide and referencedto the residual dimethyl sulphoxide signals. 1D NMR spectra, ¹H and ¹³C[APT], were acquired at 300 and 75 MHz respectively on a Varian Gemini300BB (Palo Alto Calif. USA) spectrometer. 2D NMR spectra, HSQC, HMBC,COSY and TOCSY, and a 1D NMR ¹H spectrum were acquired on a BrukerDRX600 (600 MHz) NMR spectrometer.

Analysis of NMR data was performed using ACD/SpecManager andACD/Structure Elucidator, both version 6.0 from Advanced ChemistryDevelopment, Inc. (Toronto, ON, Canada).

Electrospray Mass Sepctrometry Analysis (ES-MS)

All positive electrospray mass spectra were performed on a Finnigan/MatTSQ7000 LCMS/MS (San Jose Calif. USA).

EXAMPLE 2

EB548: Extraction and Solvent Partition

Extraction and solvent partitioning of EB548 afforded 318 g of material.Each of the extraction and solvent partition layers were tested forbioactivity using the above bioassays. It can be seen from Table 2 thatthe extracts and ethyl acetate layers of the solvent partition allcontain high CyTOX and NemaTOX activity.

TABLE 2 Activity of Extracts and Solvent Partitions. Ne Bs Tr Cy SampleTitre LD₉₉ ⁴ Titre LD₉₉ ⁴ Titre LD₉₉ ⁴ Titre LD₉₉ ⁴ EB548.MG1.20-Ext1 64120 8 990 0 256 31 EB548.MG1.20-Ext2 4 740 2 1500 1 2900 64 46EB548.MG1.28-EtOAc1 256 110 16 1800 2 14000 1024 28 EB548.MG1.28-EtOAc24 180 1 730 1 730 32 23 EB548.MG1.28-EtOAc3 0 2 150 1 290 16 18EB548.MG1.28-H2O1 2 3300 0 0 8 810 EB548.MG1.28-H2O2 2 3600 2 3600 0 32230 EB548.MG1.28-H2O3 64 110 0 4 1800 64 110 ⁴LD₉₉ in μg/mL calculatedas weight of chemical in last well with activity, however the real valuemay be lower as end point not attained.

The successive aqueous concentrated extracts were subjected to HPLC. Thecolumn used was 50×100 mm (diameter×length) packed with C18 Platinum EPS(Alltech). Approximately 2 grams of extracted material was dissolved indimethyl sulfoxide (4 mL) and subjected to preparative HPLC with typicalconditions being 60 mL/minute with gradient elution of 30% to 100%acetonitrile/water over 20 minutes followed by acetonitrile for 10minutes.

For comparison purposes the first ethyl acetate partition and the thirdwater layers were analysed by HPLC. There was little or no compounds ofinterest remaining in the third water layer of the third water/ethylacetate solvent partition.

First Preparative HPLC Fractionation

In a manner similar to that described in Phase 3 above the EB548 ethylacetate solvent partition samples where pooled and further worked upusing preparative HPLC chromatograph.

The preparative HPLC was used to produce 100 fractions. These fractionswere pooled depending on the relative concentration of compoundsindicated in the preparative HPLC chromatograph.

The bioactivity of each fraction or pooled fraction resulting from thepreparative HPLC was determined using the above bioassay method. Theresults are summarised below at Table 3.

TABLE 3 Activity of Preparative HPLC Pools. Ne Bs Tr Cy Sample Weight⁵Titre LD₉₉ ⁴ Titre LD₉₉ ⁴ Titre LD₉₉ ⁴ Titre LD₉₉ ⁴ EB548.LA3.139-1/1553.2 0 0 0 16 100 EB548.LA3.139-16 3.4 0 0 0 2 53 EB548.LA3.139-17 0.5 00 0 4 3.5 EB548.LA3.139-18 0.8 0 0 0 0 EB548.LA3.139-19 1.4 0 0 0 2 22EB548.LA3.139-20 1.9 2 30 0 0 2 30 EB548.LA3.139-21 2.8 0 0 0 2 43EB548.LA3.139-22 47.3 32 46 4 370 4 370 64 23 EB548.LA3.139-23 17.4 1634 2 270 2 270 32 17 EB548.LA3.139-24 16.0 32 16 4 130 1 500 16 31EB548.LA3.139-25 1.5 1 47 0 0 2 23 EB548.LA3.139-26/28 19.8 64 10 8 78 878 64 10 EB548.LA3.139-29 0.8 1 24 0 0 2 12 EB548.LA3.139-30/31 1.9 1 580 0 8 7.2 EB548.LA3.139-32 1.3 0 0 0 4 10 EB548.LA3.139-33/34 4.6 0 1140 0 16 8.9 EB548.LA3.139-35/36 19.7 0 0 1 620 64 10 EB548.LA3.139-3739.2 0 1 1200 4 310 128 10 EB548.LA3.139-38 43.8 0 1 1400 4 340 128 11EB548.LA3.139-39/40 148.2 4 1200 8 580 16 290 128 36 EB548.LA3.139-41/43498.9 16 970 32 490 64 240 256 61 EB548.LA3.139-44/45 9.0 0 0 0 8 35EB548.LA3.139-46/47 33.8 0 0 0 32 33 EB548.LA3.139-48/50 221.3 0 1 69002 3500 64 110 EB548.LA3.139-51/53 221.2 2 3500 0 64 110 1024 6.7EB548.LA3.139-54/55 9.2 0 2 140 1 290 16 18 EB548.LA3.139-56 24.3 0 0 1760 16 47 EB548.LA3.139-57 46.0 0 0 0 64 22 EB548.LA3.139-58/60 58.6 0 00 32 57 EB548.LA3.139-61/63 22.2 1 690 0 0 16 43 EB548.LA3.139-64 25.5 1800 0 0 32 25 EB548.LA3.139-65 7.9 1 250 0 0 8 31 EB548.LA3.139-66 4.9 277 0 0 16 10 EB548.LA3.139-67 45.3 0 0 0 4 350 EB548.LA3.139-68/69 19.30 0 0 8 75 EB548.LA3.139-70/71 5.6 0 0 0 8 22 EB548.LA3.139-72 3.2 0 0 00 EB548.LA3.139-73/75 24.8 0 0 0 2 390 EB548.LA3.139-76/78 3.1 0 0 0 0EB548.LA3.139-79 0.3 0 0 0 0 EB548.LA3.139-80/89 2.0 0 0 0 1 63EB548.LA3.139-90/100 8.0 0 0 0 4 63 ⁴LD₉₉ in μg/mL calculated as weightof chemical in last well with activity, however the real value may belower as end point not attained. ⁵Weight in mg.Second Preparative HPLC Fractionation

To prepare additional material a second preparative HPLC fraction wasperformed. The HPLC pools from the second preparative HPLC fraction didnot require bioassay as the active bands were chosen based on the UVspectra from the first preparative HPLC.

In performing the second preparative HPLC fractionation it wasdiscovered that of the maj or active bands, fractions EB548.LA3 .139-22/24, - 35/38, - 41/43 and - 51/53, the latter three showed substantialinstability. This instability was observed upon nitrogen evaporation butnot while in acetonitrile/water solution at room temperature or onvacuum evaporation. To avoid decomposition the equivalent four bands ofactive metabolites from the second preparative HPLC were individuallyback extracted in the ethyl acetate and evaporated under vacuum.Analytical HPLC of these samples confirmed minimal decomposition.

Due to overlap of Band 2 with Band 3 some of the Band 2 metabolites arein Band 3. The results of the second preparative fractionation aresummarised in Table 4.

TABLE 4 Activity of Preparative HPLC Pools. Sample Weight⁵ CommentEB548.LA4.40-1/10 14.2 EB548.LA4.40-11/13 3.0 EB548.LA4.40-14 3.0EB548.LA4.40-15/22 27.2 EB548.LA4.40-23/24 7.6 EB548.LA4.40-25/28 5.4EB548.LA4.40-29 2.6 EB548.LA4.40-30/33 8.8 EB548.LA4.40-34/35 6.5EB548.LA4.40-36/38 88.4 Band 1 - equivalent to EB548.LA3.139-22, 23, 24EB548.LA4.40-39 2.7 EB548.LA4.40-40/41 29.4 EB548.LA4.40-42 1.5EB548.LA4.40-43/44 2.1 EB548.LA4.40-45 1.6 EB548.LA4.40-46/47 27.1EB548.LA4.40-48/53 6.8 EB548.LA4.40-54/56 4.4 EB548.LA4.40-57/59 8.7EB548.LA4.40-60/61 11.1 Band 2 equivalent to EB548.LA3.139-35/36, 37, 38EB548.LA4.40-62/66 402.6 Band 3 equivalent to EB548.LA3.139-41/43EB548.LA4.40-67/73 599.3 EB548.LA4.40-74/77 25.4 Band 4 equivalent toEB548.LA3.139-51/53 EB548.LA4.40-78/79 262.1 EB548.LA4.40-80/82 328.1EB548.LA4.40-83/90 42.2 EB548.LA4.40-91/100 189.2 ⁵Weight in mg.

EXAMPLE 3

Chemical Structural Elucidation

EBI-46

The pool of like material (fractions 22 to 24 and fractions 36 to 41,80.7 mg and 120.5 mg respectively from the first and second gradientpreparative HPLC runs, respectively) was dissolved in methanol andsubjected to preparative HPLC (10 mL/min with isocratic elution of 55%water/acetonitrile over 30 minutes, through a 5 μm Phenomenex LunaC18(2) 20×100 mm column).

Fractions 17 to 20 were combined, concentrated under vacuum, freezedried and the resulting product was analysed by UV spectroscopy, HPLCanalysis, ES-MS and NMR (Table 5). From the HPLC, ES-MS and NMR analysisit was determined that EB548.LA4.61-17/20 contained the followingcompound, referred to herein as EBI-46,(12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglian-3-one):

TABLE 5 NMR Data for EBi-46 in DMSO-d₆ at 75/600 MHz. Multiplicity No. δ¹³C δ ¹H (J in Hz)  1 161.2 7.60 m  2 132.7  3 207.2  4 73.1  5 67.83.94 dd (6.5, 0.7)  6 63.6  7 63.4 3.15 s  8 34.8 2.98 d (6.6)  9 76.610 49.0 4.07 m 11 45.5 1.85 m 12 77.3 5.35 d (10.1) 13 65.3 14 35.3 1.28d (6.6) 15 26.6 16 23.4 1.14 s 17 17.0 1.20 s 18 14.5 0.68 d (6.5) 199.7 1.63 dd (2.9, 1.3) 20 62.3 3.82, 3.40 dd (12.4, 5.8), dd (12.4, 6.7) 1′ 166.9  2′ 128.1  3′ 137.1 6.72 m  4′ 14.2 1.75 m  5′ 12.1 1.74 m  1″177.3  2″ 40.4 2.30 m  3″ 25.8 1.57, 1.36 m, m  4″ 11.4 0.85 t (7.5)  5″16.1 1.04 d (7.0)  4-OH 5.49 d (0.8)  5-OH 5.17 d (6.5)  9-OH 5.29 bs20-OH 4.45 dd (6.7, 5.8)

The bioassay results of Tables 6a, 6b and 6c clearly indicate thatcompound EBI-46 has efficacy as (A) a cytotoxic agent and thereforewould be useful in the treatment and prophylaxis of cell proliferativediseases such as tumours, leukaemia, lymphoma and related disorders, (B)an antiparasitic and therefore would be useful in the treatment ofinfestation by a parasite, such as an ectoparasite and/or anendoparasites of humans and/or animals, and (C) an antibiotic andtherefore would be useful in treatment or prophylaxis of an infection bybacteria of humans and/or animals.

TABLE 6a In vitro bioassay of EBI-46 Ne Bs Tr Cy Sample Wt⁵ Titre LD₉₉ ⁴Titre LD₉₉ ⁴ Titre LD₉₉ ⁴ Titre LD₉₉ ⁴ EB548.LA4.61-17/20 109.7 2 31 150 0 — 8 7.8 ⁴LD₉₉ in pg/mL calculated as weight of chemical in lastwell with activity, however the real value may be lower as end point notattained. ⁵Wt is weight in mg.

TABLE 6b In vitro bioassays of EBI-46 Concentration of EBI-46 at whichgrowth inhibition Test cell line was observed normal human fibroblasts(NFF) None observed at 10 μg/mL mouse melanoma B16  =/<2 μg/mL humanmelanoma MM96L  =/<2 μg/mL human melanoma DO4 <100 ng/mL human melanomaMM127 <100 ng/mL Human melanoma SKMel-5 <100 ng/mL human leukemia K562<100 ng/mL human leukemia U937 <100 ng/mL human breast CF-7 <100 ng/mLMouse squamous cell carcinoma K-2 <100 ng/mL human colon Colo205 <100ng/mL human lung A549 <100 ng/mL human lung H0P62 <100 ng/mL

TABLE 6c In vivo bioassays of EBI-46 Cancer type Mode of treatmentResults Squamous Topical treatment of LK2 Resulted in cure and healingcell mouse SCC in nude mice without scarring of 3 out of 4 carcinomawith a preparation of tumours; small area of growth (SCC) EBI-46 (37ug/site) in on the 4^(th) tumour possibly due isopropanol gel once a dayto underdosing of this area. for 3 days Area subsequently healed withminimal scarring. There were no signs of systemic toxicity or lungmetastases on post mortem of experimental animals. SquamousIntralesional injection of Resulted in significant cell 21 ug EBI-46 (3× 20 ul reduction of tumour size. carcinoma injections of 7 ug each)There were no signs of (SCC) into the periphery of two systemic toxicityor lung 15-day established metastases on post mortem tumours of LK-2mouse of experimental animals. SCC in a mouse Melanoma Topical treatmentof B16 Resulted in significant melanoma tumours in inhibition of tumourgrowth. C57/B6 mice of 37 μg EBI- There were no signs of 46 per siteonce a day for systemic toxicity or lung 3 days starting at day 5,metastases on post mortem of experimental animals.EBI-47

In a similar manner to the elucidation of chemical structure outlinedabove, fractions 29 to 32 were combined, concentrated under vacuum,freeze-dried and the resulting product was analysed by UV spectroscopy,HPLC analysis, ES-MS and NMR (Table 7). From the HPLC, ES-MS and NMRanalysis it was determined that EB548.LA4.61-29/32 contained thefollowing compound, referred to herein as EBI-47(12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-tigliaen-3-one):

TABLE 7 NMR Data for EBI-47 in DMSO-d₆ at 75/600 MHz. Multiplicity No. δ¹³C δ ¹H (J in Hz)  1 161.2 7.59 m  2 132.7  3 207.2  4 73.1  5 67.83.94 d (6.5)  6 63.6  7 63.3 3.15 s  8 34.8 2.98 d (6.5)  9 76.7 10 48.94.07 m 11 45.1 1.82 m 12 76.7 5.29 d (10.3) 13 65.2 14 35.2 1.30 d (6.5)15 26.4 16 23.4 1.15 s 17 16.9 1.16 s 18 14.5 0.70 d (6.5) 19 9.7 1.64dd (2.8, 1.0) 20 62.3 3.82, 3.40 dd (12.4, 5.8), dd (12.4, 6.9)  1′175.2  2′ 40.9 2.32 m  3′ 26.2 1.55, 1.41 m  4′ 11.3 0.84 t (7.4)  5′16.6 1.06 d (7.0)  1″ 177.4  2″ 40.5 2.30 m  3″ 25.8 1.57, 1.37 m  4″11.3 0.85 t (7.4)  5″ 16.0 1.04 d (7.0)  4-OH 5.49 s  5-OH 5.17 d (6.5) 9-OH 5.30 s 20-OH 4.44 t (6.3)

The bio assay results of Tables 8a and 8b clearly indicate that compoundEBI-47 has efficacy as a (A) a cytotoxic agent and therefore would beuseful in the treatment and prophylaxis of a cell proliferative diseasessuch as a tumour, a leukaemia, a lymphoma and a related disorder, (B) anantiparasitic and therefore would be useful in the treatment ofinfestation by an antiparasitic such as an ectoparasite and/or anendoparasite of a human and/or an animal, (D) an antiprotozoal andtherefore would be useful in treatment or prophylaxis of an infection byprotozoa of humans and/or animals, and (C) an insecticide and thereforewould be useful use in the eradication and/or growth inhibition of a aninsect including a broad range of insect species.

TABLE 8a Bioassay of EBI-47 Ne BS Tr Cy Gi Titre/ Titre/ Titre/ Titre/Titre/ Sample Wt LD₉₉ ⁴ LD₉₉ ⁴ LD₉₉ ⁴ LD₉₉ ⁴ DipH⁴ LD₉₉ ⁴EB548.LA4.61-29/32 44.6 2/31 0/— 0/— 4/16 P 2/31 ⁴LD₉₉ in μg/mlcalculated as weight of chemical in last well with activity, however thereal value may be lower as end point not attained. ⁵Wt is weight in mg.

TABLE 8b Bioassay of EBI-47 Concentration of EB548.LA4.61-29/32 At whichgrowth inhibition Test cell line was observed (μg/mL) normal humanfibroblasts (NFF) None observed at 10 human leukemia K562 0.1 humanmelanoma MM418c5 0.03 human prostate DU145 10 human breast MCF-7 0.03

EBI-59

Approximately a quarter (100 mg) of pool of fractions 62 to 66, from thesecond gradient preparative HPLC run, was dissolved in methanol andsubjected to preparative HPLC (10 mL/min with isocratic elution of 80%water/acetonitrile over 25 minutes, through a 5 μm Phenomenex LunaC18(2) 20×100 mm column). Fraction 14 of the preparative HPLC wasconcentrated under vacuum, freeze-dried and the resulting product wasanalysed by UV spectroscopy, HPLC analysis, ES-MS and NMR (Table 9).From the HPLC, ES-MS and NMR analysis it was determined thatEB548.LA4.85-14 contained the following compound, referred to herein asEBI-5912-(dodeca-2,4,6-trienoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-59).

TABLE 9 NMR Data for EBI-59 in DMSO-d₆ at 75/600 MHz. Multiplicity No. δ¹³C δ ¹H (J in Hz)  1 161.3 7.60 s  2 132.7  3 207.3  4 73.1  5 67.83.94 d (6.4)  6 63.6  7 63.4 3.15 s  8 34.8 2.98 d (6.6)  9 76.6 10 49.04.07 m 11 45.5 1.85 m 12 77.1 5.34 m 13 65.3 14 35.3 1.28 m 15 26.7 1623.4 1.14 s 17 17.0 1.20 s 18 14.5 0.70 d (6.4) 19 9.7 1.64 m 20 62.33.82, 3.40 dd (12.4, 5.6), dd (12.4, 6.7)  1′ 166.0  2′ 119.9 5.89 d(15.2)  3′ 144.9 7.18 dd (15.2, 11.3)  4′ 127.8 6.33 dd (14.9, 11.3)  5′141.6 6.70 d (14.9, 10.7)  6′ 130.0 6.18 dd (15.1, 10.7)  7′ 140.6 5.98m  8′ 32.3 2.11 m  9′ 28.1 1.36 m 10′ 30.8 1.24 m 11′ 21.9 1.25 m 12′13.9 0.84 t (7.0)  1″ 177.3  2″ 40.4 2.29 m  3″ 25.8 1.57, 1.35 m, m  4″11.4 0.85 t (7.4)  5″ 16.2 1.04 d (7.0)  4-OH 5.50 s  5-OH 5.16 d (6.4)20-OH 4.45 t (6.1)

The bioassay results of Table 10 clearly indicate that compound EBI-59has efficacy as (A) a cytotoxic agent and therefore would be useful inthe treatment and prophylaxis of cell proliferative diseases such astumours, leukaemia, lymphoma and related disorders, and (B) aninsecticide and therefore would be useful use in the eradication and/orgrowth inhibition of an insect including a broad range of insectspecies.

TABLE 10 Bioassay of EBI-59 Ne Bs Tr Cy Titre/ Titre/ Titre/ Titre/DipP⁴/ Sample Wt⁵ LD₉₉ ⁴ LD₉₉ ⁴ LD₉₉ ⁴ LD₉₉ ⁴ DipH⁴ EB548.LA4.85- 19.10/— 0/— 0/— 16/3.9 A/A 14 ⁴LD₉₉ in μg/mL calculated as weight ofchemical in last well with activity, however the real value may be loweras end point not attained. ⁵Wt is weight in mg.EBI-61

In a similar manner to the elucidation of chemical structure outlinedabove the pool of fractions 35 to 36 (19.7 mg), from the second gradientpreparative HPLC run, was dissolved in methanol and subjected topreparative HPLC (10 mL/min with isocratic elution of 80%water/acetonitrile over 20 minutes, through a 5 μm Phenomenex LunaC18(2) 20×100 mm column). Fraction 8 of the preparative HPLC run wasconcentrated under vacuum, freeze-dried and the resulting product wasanalysed by UV spectroscopy, HPLC analysis, ES-MS and NMR (Table 11).From the HPLC, ES-MS and NMR analysis it was determined thatEB548.LA4.87-8 contained the following compound, referred to herein asEBI-6112-(deca-2,4-dienoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one(EBI-61).

TABLE 11 NMR Data for EBI-61 in DMSO-d6 at 75/600 MHz. Multiplicity No.δ ¹³C δ ¹H (J in Hz)  1 161.2 7.60 s  2 132.7  3 207.2  4 73.1  5 67.83.94 d (6.5)  6 63.6  7 63.4 3.16 s  8 34.9 2.96 d (6.7)  9 76.6 10 49.14.07 m 11 45.6 1.84 m 12 77.4 5.34 d (10.1) 13 65.3 14 35.3 1.29 m 1526.8 16 23.4 1.14 s 17 17.0 1.21 s 18 14.5 0.70 d (6.4) 19 9.7 1.64 dd(2.8, 1.2) 20 62.3 3.82, 3.40 dd (12.4, 5.7), dd (12.4, 6.8)  1′ 166.0 2′ 121.4 5.94 d (15.2)  3′ 139.0 7.45 ddd (15.2, 11.7, 1.0)  4′ 126.46.22 m  5′ 141.4 5.92 m  6′ 27.5 2.23 m  7′ 28.3 1.37 m  8′ 30.7 1.24 m 9′ 21.8 1.25 m 10′ 13.8 0.84 t (7.2)  1″ 177.2  2″ 40.4 2.30 m  3″ 25.81.57, 1.35 m, m  4″ 11.4 0.85 t (7.3)  5″ 16.2 1.04 d (7.1)

The bioassay results of Table 12 clearly indicate that compound EBI-61has efficacy as a (A) a cytotoxic agent and therefore would be useful inthe treatment and prophylaxis of a cell proliferative diseases such as atumour, a leukaemia, a lymphoma and a related disorder, (B) anantiparasitic and therefore would be useful in the treatment ofinfestation by an antiparasite such as an ectoparasite and/or anendoparasite of a human and/or an animal, and (C) an insecticide andtherefore would be useful use in the eradication and/or growthinhibition of an insect including a broad range of insect species.

TABLE 12 Bioassay of EBI-61 Ne Bs Tr Cy Titre/ Titre/ Titre/ Titre/DipP⁴/ Sample Wt⁵ LD₉₉ ⁴ LD₉₉ ⁴ LD₉₉ ⁴ LD₉₉ ⁴ DipH⁴ EB548.LA4.87-8 5.08/7.8 0/— 0/— 16/3.9 P/P ⁴LD₉₉ in μg/mL calculated as weight of chemicalin last well with activity, however the real value may be lower as endpoint not attained. ⁵Wt is weight in mg.

12,13-di-(2-methylbutanoyl)-1,2-2H-1,2,6,7-diepoxy-6-carboxy-4,5,9,12,13-pentahydroxy-tigliaen-3-one

EBI-47 (1 mg) in 200 μL acetone was treated with 50 μL DMDO solution andstirred for 1 hour at 0° C. and 3 hours at room temperature to give12,13-di-(2-methylbutanoyl)-1,2-2H-1,2,6,7-diepoxy-6-carboxy-4,5,9,12,13-pentahydroxy-tigliaen-3-one.MS (ESI) 617 (M+Na).

12,13-di-(2-methylbutanoyl)-5,20-di-acetoyl-4,5,9,12,13,20-hexahydroxy-tigliaen-3-one

EBI-47 (1 mg) was treated with 400 μL acetic anhydride and pyridinesolution (1:1) and stirred for 17 hours at room temperature to give12,13-di-(2-methylbutanoyl)-5,20-di-acetoyl-4,5,9,12,13,20-hexahydroxy-tigliaen-3-one. MS (ESI) 671 (M+Na).

EXAMPLE 4

Mode of Activity

While not wanting to be bound by any one theory the compound EBI-46 isconsidered to be a protein kinase C activator on the basis of it'ssimilar in vitro growth inhibition profiles to phorbol ester (TPA),selective inhibition of the growth of K562 leukemia, MCF-7 breastcancer, Colo-205 colon cancer and D04 and SKMel-5 melanoma at dosesapproximately 100-fold lower than for other human tumour cell lines andfor normal human fibroblasts. In addition, scattering of MCF-7 and HT-29colon tumour cells was observed, typical of PKC activators. A particularhuman melanoma cell line MNI96L undergoes a characteristic change tobipolar morphology when treated with TPA or with EBI-46. Additionally,given their similarity in structure, EBI-47, EBI-59 and EBI-61 are alsoconsidered to be protein kinase C activators.

EXAMPLE 5

Cytotoxicity and Reduction in Tumours With EBI-46

Materials and Methods

Isolation of EBI-46 and Related Compounds

The raw plant material of Fontainea picrosperma (EB548 or EB610) waschopped, extracted with methanol, and partitioned between water and anorganic solvent (diethyl ether or ethyl acetate). Pilot studies includedHPLC and HPTLC activity-guided analysis, then optimization of theconditions for purification of bioactivity, and confirmation ofstability. Fractionation of the bulk was conducted on silica gel 60 inpetroleum spirit/acetone/methanol or in petroleum spirit/ethylacetate/methanol, followed by HPLC. The latter involved reverse phaseseparation on a C18 Phenomenex Lunar 5 micron, 250×4.6 mm column inmethanol-water.

The following gradient (Method EB.M, flow 2 mL/min) was used for theseparation reported in Table 12.

TABLE 12 Time 0 15 39 40 45 46 55 % MeOH 70 80 84 100 100 70 70

Bioactivity Assays

The human tumour cell lines were: MCF-7 and T47D, breast cancer; DU145and PC_(3,) prostate cancer; CI80-13S, ovarian cancer; MM96L, D04,SkMel5, melanoma; HT29, colon cancer. Neonatal foreskin fibroblasts(NFF) were used as normal control cells. Cells were cultured at 37° C.in 5% carbon dioxide/air, in RPMI 1640 medium containing 10% fetal calfserum.

For growth inhibition assays, cells (2000-5000/well) were seeded in96-well plates in duplicate, treated with drug next day and allowed togrow for a further 5-6 days. The plates were then washed in PBS(phosphate buffered saline, pH 7.2), fixed with ethanol and stained byaddition of 50 μL 0.4% SRB (sulforhodamine) in 1% acetic acid. The platewas left at room temperature for a minimum of 15 minutes, then washedrapidly with tap water and then twice with 1% acetic acid. Afteraddition of 100 μL/well of 10 mM Tris base (unbuffered, pH>9), plateswere left for a minimum of 5 minutes, then the absorbance was read at564 nm on the ELISA reader, with a 3 second prior shaking. Aftersubtraction of a blank (wells with no cells, typically about 0.04),growth inhibition was calculated as % of the untreated control andplotted against dose.

Animal experiments were conducted under Queensland Institute of MedicalResearch (QIMR) Animal Ethics Committee approvals. The B16 mousemelanoma model was obtained by injecting 0.5 million B16 mouse melanomacells subcutaneously into each of the 2 flanks of a male C57BL/6 mouse.The human tumour xenografts were obtained by injecting 2 million of therespective tumour cell line into each of 4 sites on the flanks of malenude mice (BALB/c background). EBI-46 was applied: (a) topically bydilution of a concentrated solution in acetone into an isopropanol gel,once a day for 3 days; (b) intralesionally by injection into the tumourof drug dissolved in 25% propylene glycol-0.1 M saline, (c) systemicallyby injection intraperitoneally in 25% propylene glycol in 0.1 M saline.Tumour size was measured with electronic callipers in mm and convertedto volume (cubic mm) using the formula:Tumour volume=length×breadth×breadth/2.Results

1. Purification of EBI-46 and Related Compounds

The organic extract was fractionated by chromatography on silica givinga fraction (548-35) containing a bioactive peak of high purity (RT:25.131 minutes). Further purification by HPLC yielded >2 g of EBI-46(RT: 25.262 minutes), from 2 kg of plant material.

2. Purity, Stability and Solubility

The bulk sample of EBI-46 was found to be >95% pure by UV and NMR, thelimit of detection of the instruments.

Retention of bioactivity through extraction and chromatography stepsimplied that the structure was stable, and this has been confirmed tothe extent that solutions of EBI-46 in ethanol retain bioactivity whenheld at 4° C. for 4 weeks. This was confirmed by an HPLC study ofstability in the preferred delivery vehicle for intralesional injection(PEG 400 containing 10% ethanol) held at 37° C. The structure has noreadily reactive groups which might otherwise confer instability.

Being a diterpene ester, EBI-46 is highly soluble in organic solventsincluding biocompatible solvents such as acetone, alcohols and PEG 400.It requires a small amount of such solvents to form aqueous solutions.Solubility tests have demonstrated 100% solubility at all 3concentrations tested so far: 450 μg/mL in 90% water, 50 μg/mL in 99%water and 5 μg/mL in 99.9% water. Higher solubilities may well beachieveable.

Note that EBI-46 is a potent drug and only small amounts are required.Thus for intralesional injection of EBI-46, the concentration was 400μg/mL (and no water was required in this case).

3. Bioactivity Profile of EBI-46 and Related Compounds In Vitro: Arrestof Cell Growth

The ability of the EB548 crude extract (Table 13), and purified EBI-46(FIG. 4) to block the growth of cultured human tumour cell lines and anormal strain (human diploid fibroblasts) was tested in aclonogenic-type assay where many generations were allowed to elapse (5-6days treatment) before measuring cell growth (Sulfurhodamine proteinstain). Changes in morphology were also scored, and these were identicalto those induced by the known protein kinase C (PKC) activator TPA(tetradecanoyl phorbol acetate), namely extreme bipolar morphology inthe MM96L cell line and scattering of the MCF-7 cell clusters.

TABLE 13 Relative sensitivities of human cells to growth inhibition byEBI-46-rich crude extract (EB548) Numbers represent the amount of crudeextract required to produce 50% growth inhibition (IC50) of the cellline, relative to the JAM cell line (arbitrarily set to 100). Cell EB548Normal NFF 700 PBMC 1.2 Solid tumour A549 lung <0.01 D04 melanoma 0.003HOP62 lung 0.002 SKMel-28 melanoma 200 MM96L melanoma 60 MM127 melanoma<0.001 MCF-7 breast 0.005 Colo205 colon <0.001 HT29 colon 200 JAMovarian 100 CI80-13S ovarian 200 Leukemia K562 <0.001 U937 <0.01These results were confirmed for purified EBI-46 (FIG. 4)

The EBI-46/EB548-sensitive cell lines (K562, HL60, MCF7, Colo205, MM127,D04, U937) were also sensitive in a similar degree to growth inhibitionby TPA (results not shown), at doses approximately 100-fold lower thanfor other human tumour cell lines and for normal human fibroblasts andPBMC (peripheral blood mononuclear cells). Further, EBI-46 induced thesame bipolar morphology in MM96L and cell scattering of MCF-7 as TPA.Since such effects have previous been shown to be blocked by a PKCinhibitor, EBI-46 is considered to be a PKC activator. Similarbioactivities were observed for EBI-47, EBI-61, EB610 p4 011206 andEBI-59 as set out in Table 14.

TABLE 14 HPLC of Fontainea picrosperma isolate under followingconditions: Column: Phenomenex Luna 5u 250 × 4.60 mm C18 Flow: 2mL/minute Solvent system: Methanol/water Gradient: Time (min) 0 15 39 4045 46 55 % MeOH 70 80 84 100 100 70 70 Retention Time PKC Compounddescriptor (min) activity* Structure EBI-46 23.2 yes EBI-46 EBI-49 22.6#yes EBI-49 EB610_EB_49.1 49.1 yes phorbol ring system*** EB610_EB_26.626.6 yes EBI-47 EB610_EB_32.3 32.3 yes phorbol ring system***EB610_EB_48.2 48.2 yes EBI-61 EB610_p4_011206 46.4 yes phorbol ringsystem*** EBI-59 17.8** yes EBI-59 *Activates PKC in cultured tumourcells as defined by scattering and growth inhibition of MCF7 breastcancer cells **Run on a C18 Luna column, isocratic 80%acetonitrile-water ***As defined by NMR #Run on a different HPLC column.

These compounds are therefore also considered to be PKC activators andof potential utility in the same indications as demonstrated for EBI-46below.

4. Efficacy of EBI-46 in Treatment of Subcutaneous Tumours in Mice:Topical Application

Topical application of EBI-46 in an isopropanol gel was carried out onthe aggressive B16 mouse melanoma in its natural (syngeneic) host,C57BL/6 mice (0.5 million tumour cells injected per site).

The frequency (1 daily dose for 3 days only) and dose level for topicalapplication of PKC activators was selected on the basis of in vitroactivity on cell lines. The materials were dissolved in acetone anddiluted into an isopropanol gel for topical application.

The aggressive and rapidly growing B16 mouse melanoma is recognised as avery stringent tumour model in which to test anticancer agents.Stringency was further increased by injecting at least 10× more tumourcells than the minimum required to form a tumour in the animal. Aconfounding factor in determining the efficacy of topical treatments wasthat some tumour cells escaped from the subcutaneous site at a earlystage and became established in the underlying muscle where it isassumed that the drug and its associated dermal host response did notreach. Such tumours could be distinguished from subcutaneous tumours bytheir immobility when the skin was pulled around the body of the animal.

It was therefore highly significant that approximately 150 μgEBI-46/site gave a good response (4 mice and 4 controls, 2 sites/mouse),with one site apparently cured but the mouse had to be euthanasedbecause the other site was growing. The inflammatory response was mild.There was no sign of systemic toxicity or lung metastases with any ofthe drugs.

The regrowth of tumour cells after 10 days was not surprising, given thestringency of the model (FIG. 5) and the somewhat arbitrary choice ofdose and regimen.

The above was repeated using the UVB-induced mouse squamous cellcarcinoma (SCC) grown on nude mice (FIG. 6) and treated withpartially-purified EBI-46. This more realistic model for skin cancersshowed an extremely high response rate which was maintained over a longperiod.

Again, an excellent result was obtained, with relapses occurring after 6weeks due primarily to outgrowth of tumours from the underlying muscle,presumably due to being out of reach of the topical drug.

The most recent experiment with topical application used 2 differentdoses of EBI-46 (FIG. 7). The low dose was 100 μg/site and the high dosewas 350 μg/site/treatment.

This experiment gave an excellent result at 350 μg EBI-46/site, andshowed that it was important to achieve a certain dose level to achieveefficacy.

5. Efficacy of EBI-46 in Treatment of Subcutaneous Tumours in Mice:Intralesional Injection

A pilot study was conducted on 6 mm×6 mm LK-2 tumours established innude mice. Approximately 50 μL of a solution of EB548 fractions(approximately 20 μg EBI-46 in the EB548-35 fraction) in salinecontaining 20% acetone were injected in 3 sites around the periphery ofeach lesion. This was only done once.

The result showed rapid ablation of visible tumours, (FIG. 8) and aninflammatory response at the site of injection. Tumour growth eventuallyrecovered, presumably due to non-optimal delivery.

The above procedure was then modified by using PEG 400 containing 10%ethanol as the vehicle. EBI-46 is freely soluble in this mixture and theincreased viscosity served to restrict delivery of drug to the tumoursite.

With PEG 400 delivery, 10 μg EBI-46 in 25 μL solution was injected (29gauge) with a 0.5 mL insulin needle into a 7 mm×7 mm tumour, highlyvisible on the left flank of a nude mouse.

By 16 hours, a marked inflamed area had developed and the tumour lumphad largely gone. A small area of normal skin on the ridge of the backwas accidentally treated topically with the preparation, and developed amild inflammatory response.

Seven days later, the tumour site was still flat and a scab had formed.The normal treated skin on the ridge of the back also formed a scab.This mouse remained tumour-free for over 9 months and was finallyeuthanased due to an unrelated condition (swollen penis).

In addition to the advantage of viscosity for localization of drug, PEG400 gave fewer problems with leaking out after withdrawal of the needle.PEG 400 alone had no effect when injected into an LK2 tumour on anothermouse.

A second mouse model was tested in a pilot study, involving an 8 mmdiameter human nasopharyngeal tumour implanted and growingsubcutaneously on the neck of a SCID-NOD mouse. Up to 3 injections ofEBI-46 (total of 25 μg in 75 μL25% propylene glycol-saline) were madeinto the NPC tumours of 2 mice. The scab sloughed off the treated sitein one mouse, with no sign of residual tumour. Growth of the tumour inthe second mouse was delayed but not ablated.

Systemic Administration of EBI-46

A variety of reports using cultured cells suggest that PKC activatorsmay have potential for the treatment of lymphoid neoplasms. The murineB-cell lymphoma line A20 was used as an experimental model because ithas been reported to grow well in mice and closely models the humansituation.

SCID-NOD mice (BALB/c background) were shaved and 10E7 A20 cellsinjected subcutaneously (2 sites per mouse). The tumours tended to growin a flat, diffuse manner and became raised and measurable at the 10E7sites only after about 15 days. One mouse with 10E7 tumours was theninjected intraperitoneally from day 18 with a total of 5 doses of 20-25μg EBI-46 in 25% propylene glycol-saline. The solution was stable forweeks at 4° C., and there was no sign of insolubility at thisconcentration (250 μg/mL).

The results suggest that tumour growth in the injected mouse wasstrongly inhibited by EBI-46, compared with an untreated 10E7 mouse.Growth increased when the treatment stopped and the mouse was euthanasedat 27 days.

Delivery Vehicles for EBI-46

Consideration of the structure and stability of EBI-46 leads to the useof protic solvents that are biocompatible. Benzyl alcohol and Cremaphorwould be possibilities but have not been tested. PEG 400 was chosenbecause of its common usage, but similar solvents could well besuitable; and the use of larger needles would obviate the need to diluteslightly with ethanol. None of the above materials were deliberatelysterilized, despite being used in the immunocompromised nude mice.

Safety Issues

The operator, as with any potent drug, should wear personal protection(gloves, coat/gown, eye protection). EBI-46 can cause inflammation ofskin. It can be deactivated with sodium carbonate solution.

The animals have shown no weight loss, signs of distress or sideeffects. Internal organs appeared to be normal on dissection but nohistology or formal toxicology has been done.

Discussion

EBI-46 is solvent extractable from EB548 material and although othercompounds with similar activity are present, EBI-46 travels in anuncluttered region of the chromatogram and therefore is relatively easyto purify. The same bioactivities were obtained from the crude extractthrough to the purified structure. Properties relevant to its potentialuse as a pharmaceutical have so far been favourable: availability,purity, stability and solubility in delivery vehicle.

The cell growth inhibition profile revealed EBI-46 to be a PKCactivator, showing very high selectivity for a subset of solid tumourand leukemia cell lines, compared with normal cells and some othertumour cell lines. Local application such as topical cream orintralesional injection into lesions is likely to clear these sitesbecause of a combination of direct killing (high local dose) andelimination of peripheral tumour cells by the host's innate immuneresponse as evidenced by the early inflammatory reaction at the site ofapplication.

Intralesional injection required less drug than topical application, andonly one treatment, to obtain a significant response. If relapse occurs,for example on one side of the original lesion, repeated injectionswould be possible. Injection also provides a more positive delivery thanrelying on topical application on sites of different skin thickness.

It is important to note that efficacy of local treatment does notrequire the target tumour to be intrinsically sensitive to EBI-46. Theaggressive B16 mouse melanoma cells for example are quite resistant inculture but respond to the drug topically in the mouse. Presumably, thevigorous host response is a major factor. This C57BL/6 strain is verydifferent from the BALB/c background of the nude mouse, indicating thatneither strain differences nor lack of adequate T-cell immunity inhibitthe efficacy of EBI-46.

The ability of local treatment with EBI-46 to work in species other thanmice remains to be evaluated. Mouse skin is very thin (half thethickness of human skin), thus making intralesional injection moreattractive. PEG 400 was chosen as a vehicle because it is usedextensively for drug delivery and because its viscosity (loweredslightly with 10% ethanol to achieve injectability) may limit spreadingaway to far from the injection site.

Note also that EBI-46 causes an inflammatory response in normal skin.There are anecdotal reports of various plant saps being used to treatwarts. This raises the possibility of using EBI-46 to ablate keloidscars, psoriasis, warts, proud flesh and other non-malignant conditionsof the skin.

Local treatment may find significant application beyond skin lesions. Itmay be feasible to locally treat, by injection or suitably-formulatedtopical preparations, life-threatening tumours such as those of the oralcavity, oesophagus and bowel. This could be carried out in conjunctionwith physical or pharmacological means of limiting escape of the druginto the circulation.

Throughout this specification, unless the context requires otherwise,the word “comprises”, and variations such as “comprise” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers or steps but not to the exclusion of any other integer orgroup of integers.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. It will therefore beappreciated by those of skill in the art that, in light of the instantdisclosure, various modifications and changes can be made in theparticular embodiments exemplified without departing from the scope ofthe present invention.

All computer programs, algorithms, patent and scientific literaturereferred to herein is incorporated herein by reference.

The invention claimed is:
 1. A method of reducing scar tissue in asubject comprising administering to the scar tissue a compound offormula (II) or a pharmaceutically acceptable salt thereof, wherein thecompound of formula (II) has the following structure:

wherein: X is —O—; R¹ and R² are independently selected from hydrogen,OH, OC₁-C₁₀ alkyl, OC₂-C₁₀alkenyl, OC₂-C₁₀alkynyl, Ocycloalkyl,OC(O)C₁-C₁₀alkyl, OC(O)C₂-C₁₀ alkenyl, OC(O)C₂-C₁₀alkynyl,OC(O)cycloalkyl, OC(O)NHC₁-C₁₀ alkyl, OC(O)NHC₂-C₁₀alkenyl,OC(O)NHC₂-C₁₀alkynyl, OC(O)NHcycloalkyl, OC(O)NHaryl,OC(S)NHC₁-C₁₀alkyl, OC(S)NHC₂-C₁₀alkenyl, OC(S)NHC₂-C₁₀ alkynyl,OC(S)NHcycloalkyl and OC(S)NHaryl, or R¹ and R² taken together are ═O,═S, ═NH or ═N(C₁-C₆ alkyl); R³ is hydrogen, —C₁-C₁₀alkyl, —C₂-C₁₀alkenylor —C₂-C₁₀alkynyl; R⁴ and R⁵ are each hydrogen, —C₁-C₁₀alkyl,—C₂-C₁₀alkenyl, —C₂-C₁₀ alkynyl, —OH, —OC₁-C₁₀alkyl, —OC₂-C₁₀alkenyl,—OC₂-C₁₀alkynyl, —Ocycloalkyl, —OC(O)C₁-C₁₀ alkyl, —OC(O)C₂-C₁₀alkenyl,—OC(O)C₂-C₁₀alkynyl, —OC(O)cycloalkyl, —OC(O)aryl, —OC(O)NHC₂-C₁₀ alkyl,—OC(O)NHC₂-C₁₀ alkenyl, —OC(O)NHC₂-C₁₀alkynyl, —OC(O)NHcycloalkyl,—OC(O)NHaryl, —OC(S)NHC₁-C₁₀alkyl, —OC(S)NHC₂-C₁₀ alkenyl,—OC(S)NHC₂-C₁₀alkynyl, —OC(S)NHcycloalkyl, —OC(S)NHaryl, F, Cl, Br, I,—CN, —NO₂ or N(R²⁵)₂, or R⁴ and R⁵ taken together form a double bond orare —O—, —S—, —NR²⁵— or —CR²⁶R²⁷—; R⁶ is selected from hydrogen,—C₁-C₁₀alkyl, —C₂-C₁₀alkenyl and —C₂-C₁₀ alkynyl; R⁸ is selected fromhydrogen, —C₁-C₁₀alkyl, —C₂-C₁₀alkenyl and —C₂-C₁₀ alkynyl; R¹⁰ isselected from hydrogen, —C₁-C₁₀alkyl, —C₂-C₁₀alkenyl and —C₂-C₁₀alkynyl; R¹¹ is selected from OH, OC₁-C₂₀alkyl, OC₂-C₂₀alkenyl, OC₂-C₂₀alkynyl, Ocycloalkyl, OC(O)C₁-C₂₀alkyl, OC(O)C₂-C₂₀alkenyl,OC(O)C₂-C₂₀alkynyl, OC(O)cycloalkyl, OC(O)aryl, OC(O)NHC₁-C₂₀alkyl,OC(O)NHC₂-C₂₀alkenyl, OC(O)NHC₂-C₂₀alkynyl, OC(O)NHcycloalkyl,OC(O)NHaryl, OC(S)NHC₁-C₂₀alkyl, OC(S)NHC₂-C₂₀alkenyl,OC(S)NHC₂-C₂₀alkynyl, OC(S)NHcycloalkyl and OC(S)NHaryl, or R¹⁰ and R¹¹taken together form a carbonyl group (═O); R¹² is selected from OH,OC₁-C₂₀alkyl, OC₂-C₂₀alkenyl, OC₂-C₂₀ alkynyl, Ocycloalkyl,OC(O)C₁-C₂₀alkyl, OC(O)C₂-C₂₀alkenyl, OC(O)C₂-C₂₀ alkynyl,OC(O)cycloalkyl, OC(O)aryl, OC(O)NHC₁-C₂₀alkyl, OC(O)NHC₂-C₂₀alkenyl,OC(O)NHC₂-C₂₀alkynyl, OC(O)NHcycloalkyl, OC(O)NHaryl,OC(S)NHC₁-C₂₀alkyl, OC(S)NHC₂-C₂₀alkenyl, OC(S)NHC₂-C₂₀alkynyl,OC(S)NHcycloalkyl and OC(S)NHaryl; R¹³ and R¹⁴ are independentlyselected from hydrogen and —C₁-C₁₀ alkyl; R¹⁸ is selected fromC₁-C₂₀alkyl, C₂-C₂₀alkenyl, C₂-C₂₀alkynyl, OH, OC₁-C₂₀alkyl,OC₂-C₂₀alkenyl, OC₂-C₂₀ alkynyl, Ocycloalkyl, OC(O)C₁-C₂₀alkyl,OC(O)C₂-C₂₀alkenyl, OC(O)C₂-C₂₀ alkynyl, OC(O)cycloalkyl, OC(O)aryl,NHC₁-C₂₀alkyl, NHC₂-C₂₀alkenyl, NHC₂-C₂₀alkynyl, NHcycloalkyl, NHaryl,OC(O)NHC₁-C₂₀alkyl, OC(O)NHC₂-C₂₀alkenyl, OC(O)NHC₂-C₂₀alkynyl,OC(O)NHcycloalkyl, OC(O)NHaryl, OC(S)NHC₁-C₂₀alkyl,OC(S)NHC₂-C₂₀alkenyl, OC(S)NHC₂-C₂₀alkynyl, OC(S)NHcycloalkyl andOC(S)NHaryl; R¹⁹ and R²⁰ are independently selected from hydrogen,—C₁-C₆ alkyl, —C₂-C₆ alkenyl, —C₂-C₂₀alkynyl, —OH, —OC₁-C₆alkyl,—OC₂-C₆alkenyl, —OC₂-C₆ alkynyl, —Ocycloalkyl, —OC(O)C₁-C₆alkyl,—OC(O)C₂-C₆alkenyl, —OC(O)C₂-C₆ alkynyl, —OC(O)cycloalkyl and —OC(O)arylor R¹⁹ and R²⁰ taken together form a carbonyl or thiocarbonyl group; R²¹is selected from hydrogen, —C₁-C₁₀alkyl, —C₂-C₁₀alkenyl and —C₂-C₁₀alkynyl; R²² is selected from hydrogen, OH, OC₁-C₂₀alkyl,OC₂-C₂₀alkenyl, OC₂-C₂₀alkynyl, Ocycloalkyl, OC(O)C₁-C₂₀alkyl,OC(O)C₂-C₂₀alkenyl, OC(O)C₂-C₂₀ alkynyl, OC(O)cycloalkyl, OC(O)aryl,OC(O)NHC₁-C₂₀alkyl, OC(O)NHC₂-C₂₀alkenyl, OC(O)NHC₂-C₂₀alkynyl,OC(O)NHcycloalkyl, OC(O)NHaryl, OC(S)NHC₁-C₂₀alkyl,OC(S)NHC₂-C₂₀alkenyl, OC(S)NHC₂-C₂₀ alkynyl, OC(S)NHcycloalky and,OC(S)NHaryl, or R²¹ and R²² taken together form a carbonyl group; R²³ isselected from hydrogen, OH, OC₁-C₂₀alkyl, OC₂-C₂₀alkenyl,OC₂-C₂₀alkynyl, Ocycloalkyl, OC(O)C₁-C₂₀alkyl, OC(O)C₂-C₂₀alkenyl,OC(O)C₂-C₂₀alkynyl, OC(O)cycloalkyl, OC(O)aryl,—OC(O)NHC₁-C₂₀alkyl,OC(O)NHC₂-C₂₀alkenyl, OC(O)NHC₂-C₂₀alkynyl, OC(O)NHcycloalkyl,OC(O)NHaryl, OC(S)NHC₁-C₂₀alkyl, OC(S)NHC₂-C₂₀alkenyl, OC(S)NHC₂-C₂₀alkynyl, OC(S)NHcycloalkyl and OC(S)NHaryl; R²⁴ is selected fromhydrogen, OH, OC₁-C₂₀alkyl, OC₂-C₂₀alkenyl, OC₂-C₂₀alkynyl, Ocycloalkyl,OC(O)C₁-C₂₀alkyl, OC(O)C₂-C₂₀alkenyl, OC(O)C₂-C₂₀ alkynyl,OC(O)cycloalkyl, —OC(O)aryl, OC(O)NHC₁-C₂₀alkyl, OC(O)NHC₂-C₂₀alkenyl,OC(O)NHC₂-C₂₀alkynyl, OC(O)NHcycloalkyl, OC(O)NHaryl,OC(S)NHC₁-C₂₀alkyl, OC(S)NHC₂-C₂₀alkenyl, OC(S)NHC₂-C₂₀alkynyl,OC(S)NHcycloalkyl and OC(S)NHaryl; R²⁵ is selected from hydrogen and—C₁-C₁₀alkyl; R²⁶ and R²⁷ are independently selected from hydrogen,—C₁-C₁₀alkyl, —OH, —OC₁-C₁₀ alkyl; wherein each alkyl, alkenyl, alkynyl,cycloalkyl and aryl is optionally substituted with one or more optionalsubstituents, wherein each optional substituent is independently —F,—Cl, —Br, —I, —CN, —CF₃, —CO₂R²⁸, —COR²⁸, —SR²⁸, —N(R²⁸)₂, —NO₂,—NR²⁸OR²⁸, —ON(R²⁸)₂, —SOR²⁸, —SO₂R²⁸, —SO₃R²⁸, —SON(R²⁸)₂, —SO₃N(R²⁸)₂,—P(R²⁸)₃, —P(═O)(R²⁸)₃, —OSi(R²⁸)₃, —OB(R²⁸)₂ and wherein R²⁸ ishydrogen, —C₁-C₆ alkyl, —C₂-C₆alkenyl, —C₂-C₆alkynyl, C₈cycloalkyl,—C₆-C₁₄aryl, arylalkyl, —C₁-C₆ haloalkyl, —C₁-C₆dihaloalkyl or—C₁-C₆trihaloalkyl; or a geometric isomer or stereoisomer thereof or apharmaceutically acceptable salt thereof.
 2. A method according to claim1 wherein the compound of formula (II) is a compound of formula (III):

wherein: R³ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl; R⁴ and R⁵ areeach independently hydrogen, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —OH, —OC₁-C₆alkyl, —OC₂-C₆ alkenyl, —OC(O)C₁-C₆ alkyl, —OC(O)C₂-C₆ alkenyl,—OC(O)aryl, F, Cl, Br or I, or R⁴ and R⁵ taken together form a doublebond or are —O—; R⁶ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl; R⁸ ishydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl; R¹³ and R″ are eachindependently hydrogen or —C₁-C₆ alkyl; R^(a) is hydrogen, —C₁-C₂₀alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, —C(O)C₂-C₂₀ alkyl, —C(O)C₂-C₂₀alkenyl, —C(O)C₂-C₂₀ alkynyl, —C(O)cycloalkyl, —C(O)aryl, —C(O)NHC₁-C₂₀alkyl, —C(O)NHC₂-C₂₀ alkenyl, —C(O)NHC₂-C₂₀ alkynyl, —C(O)NHcycloalkyl,—C(O)NHaryl, —C(S)NHC₁-C₂₀ alkyl, —C(S)NHC₂-C₂₀ alkenyl, —C(S)NHC₂-C₂₀alkynyl, —C(S)NHcycloalkyl and —C(S)NHaryl; R^(b), R^(c), R^(d), R^(e)and R^(f) are each independently selected from the group consisting ofhydrogen, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl, —C₂-C₂₀ alkynyl, —C(O)C₁-C₂₀alkyl, —C(O)C₂-C₂₀ alkenyl, —C(O)C₂-C₂₀ alkynyl, —C(O)cycloalkyl,—C(O)aryl, —C(O)NHC₁-C₂₀ alkyl, —C(O)NHC₂-C₂₀ alkenyl, —C(O)NHC₂-C₂₀alkynyl, —C(O)NHcycloalkyl, —C(O)NHaryl, —C(S)NHC₁-C₂₀ alkyl,—C(S)NHC₂-C₂₀ alkenyl, —C(S)NHC₂-C₂₀ alkynyl, —C(S)NHcycloalkyl and—C(S)NHaryl; wherein each alkyl, alkenyl and aryl group is optionallysubstituted with one or more optional substituents wherein each optionalsubstituent is as defined in claim 1; or a geometric isomer orstereoisomer or a pharmaceutically acceptable salt thereof.
 3. Themethod according to claim 1 wherein the compound of formula (II) is acompound of formula (IV):

wherein: R³ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl; R⁴ and R⁵ areeach independently hydrogen, —C₁-C₆ alkyl, —C₂-C₆ alkenyl, —OH, —OC₁-C₆alkyl, —OC₂-C₆ alkenyl, —OC(O)C₁-C₆ alkyl, —OC(O)C₂-C₆ alkenyl,—OC(O)aryl, F, Cl, Br or I, or R⁴ and R⁵ taken together form a doublebond or are —O—; R⁶ is hydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl; R⁸ ishydrogen, —C₁-C₆ alkyl or —C₂-C₆ alkenyl; R¹⁰ is hydrogen; R¹¹ ishydroxy, —OC₁-C₂₀ alkyl, —OC₂-C₂₀ alkenyl, —OC(O)C₂-C₂₀ alkyl,—OC(O)C₂-C₂₀ alkenyl or —OC(O)aryl; or R¹⁰ and R¹¹ taken together form acarbonyl group; R^(12′) is hydrogen, —C₁-C₂₀ alkyl, —C₂-C₂₀ alkenyl,—C(O)C₁-C₂₀ alkyl, —C(O)C₂-C₂₀ alkenyl or —C(O)aryl; R¹³ and R¹⁴ areeach independently hydrogen or —C₁-C₆ alkyl; R^(18′) is hydrogen, —C₁-C₆alkyl, —C₂-C₆ alkenyl, —C(O)C₁-C₆ alkyl, —C(O)C₂-C₆ alkenyl or—C(O)aryl; R¹⁹ and R²⁰ are independently selected from the groupconsisting of hydrogen, —OH, —OC₁-C₆ alkyl, —OC₂-C₆ alkenyl or R¹⁹ andR²⁰ taken together form a carbonyl group; R²¹ is hydrogen; R²² ishydroxy, —OC₁-C₆ alkyl, —OC₂-C₆ alkenyl, —OC(O)C₁-C₆ alkyl, —OC(O)C₂-C₆alkenyl, —OC(O)aryl or R²¹ and R²² taken together form a carbonyl group;wherein each alkyl, alkenyl and aryl is optionally substituted with oneor more optional substituents, wherein each optional substituent is asdefined in claim 1; or a geometric isomer or stereoisomer thereof or apharmaceutically acceptable salt thereof.
 4. The method according toclaim 1 wherein R¹ and R² are independently selected from the groupconsisting of hydrogen, hydroxy, —OC₁-C₆alkyl, —OC₂-C₆alkenyl,—OC(O)C₁-C₆ alkyl or —OC(O)C₂-C₆alkenyl or R¹ and R² taken together forma carbonyl group.
 5. The method according to claim 1 wherein R⁴ and R⁵form a double bond or R⁴ and R⁵ are hydrogen or together are —O—.
 6. Themethod according to claim 1 wherein R⁸ is hydrogen or —C₁-C₃ alkyl. 7.The method according to claim 1 wherein R¹¹ is selected from the groupconsisting of hydroxy, —C₁-C₂₀alkyloxy, —C₂-C₂₀alkenyloxy,—OC(O)C₂-C₂₀alkyl or —OC(O)C₂-C₂₀ alkenyl and R¹² is selected fromhydroxy, —C₁-C₂₀alkyloxy, —C₂-C₂₀alkenyloxy, —OC(O)C₁-C₂₀alkyl or—OC(O)C₂-C₂₀alkenyl.
 8. The method according to claim 1 wherein R¹³ andR¹⁴ are independently selected from the group consisting of hydrogen and-C₁-C₃alkyl.
 9. The method according to claim 1 wherein R¹⁹ and R²⁰ areindependently hydrogen, hydroxy, —OC₁-C₃alkyl and —OC₂-C₃alkenyl or R¹⁹and R²⁰ taken together form a carbonyl group and R¹⁸ is selected fromthe group consisting of hydroxy, —OC₁-C₆ alkyl, —OC₂-C₆alkenyl,—OC(O)C₁-C₆alkyl and —OC(O)C₂-C₆alkenyl.
 10. The method according toclaim 1 wherein R²² is hydroxy, —OC₁-C₆alkyl, —OC₂-C₆alkenyl,—OC(O)C₁-C₆alkyl or —OC(O)C₂-C₆alkenyl.
 11. The method according toclaim 1 wherein R²³ and R²⁴ are each hydroxyl.
 12. The method accordingto claim 1 wherein the compound is selected from:12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one;12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-tigliaen-3-one;12-(dodeca-2,4,6-trienoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one;12-(deca-2,4-dienoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one;12,13-di-(2-methylbutanoyl)-1,2-2H-1,2,6,7-diepoxy-6-carboxy-4,5,9,12,13-pentahydroxy-tigliaen-3-one;and12,13-di-(2-methylbutanoyl)-5,20-di-acetoyl-4,5,9,12,13,20-hexahydroxy-tigliaen-3-one;or a pharmaceutically acceptable salt thereof.
 13. The method accordingto claim 1 wherein the scar tissue is a keloid.
 14. The method accordingto claim 1 wherein the compound of formula (II) or a pharmaceuticallyacceptable salt thereof is administered to the scar tissue topically.15. The method according to claim 1 wherein the compound of formula (II)or a pharmaceutically acceptable salt thereof is administered to thescar tissue intra-lesionally.
 16. The method according to claim 13wherein the compound of formula (II) or a pharmaceutically acceptablesalt thereof is administered to the keloid topically.
 17. The methodaccording to claim 13 wherein the compound of formula (II) or apharmaceutically acceptable salt thereof is administered to the keloidintra-lesionally.
 18. The method according to claim 13 wherein thecompound of formula (II) is12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one.19. The method according to claim 16 wherein the compound of formula(II) is12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one.20. The method according to claim 17 wherein the compound of formula(II) is12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one.